The sequence element of the internal ribosome entry site and a 25-kilodalton cellular protein contribute to efficient internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by internal entry of ribosomes into the 5' noncoding region (NCR). This process depends on genomic elements within the 5' NCR called the internal ribosome entry site (IRES) and may involve host factors. The alpha-branch structure (nucleotides 47 to 67) of the HCV IRES is considered a cis-acting element critical for translation initiation because it is indispensable for translation in vitro (S. Fukushi, K. Katayama, C. Kurihara, N. Ishiyama, F. B. Hoshino, T. Ando, and A. Oya, Biochem. Biophys. Res. Commun. 199:425-432, 1994). In order to further characterize the function of the alpha-branch, we determined whether sequence exchange within the alpha-branch had any effect on translation initiation. An in vitro translation study revealed that the stem sequences of this region played an important role in efficient IRES function. In addition to several HeLa cell proteins, which had a binding affinity for the 5' NCR, a novel 25-kDa protein that specifically interacted with the HCV IRES was discovered. The binding affinity of the 25-kDa protein for the 5' NCR was correlated with the efficiency of translation initiation of HCV RNA, indicating a critical role for the 25-kDa protein in HCV translation.     

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.     

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.     

Interaction of Poly(rC) Binding Protein 2 with the 5′ Noncoding Region of Hepatitis A Virus RNA and Its Effects on Translation

Utilization of internal ribosome entry segment (IRES) structures in the 5′ noncoding region (5′NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5′NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5′NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5′-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.     

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. We have previously shown that a structural motif within the foot-and-mouth-disease virus IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in cultured cells, leading to inhibition of translation. Inhibition of IRES activity was dependent on the expression of the active ribozyme RNA subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. RNAs carrying defective IRES mutants that were substrates of the ribozyme in vivo revealed an increased translation of the reporter in response to the expression of the active ribozyme. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 5′-end-dependent translation of RNase P target RNAs. We conclude that the IRES element is inactivated by expression in cis of RNase P in the cytoplasm of cultured cells, providing a promising antiviral tool to combat picornavirus infections. Furthermore, our results reinforce the essential role of the structural motif that serves as RNase P recognition motif for IRES activity.     

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

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

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region.

Translation of the human hepatitis C virus (HCV) RNA genome occurs by a mechanism known as "internal ribosome entry." This unusual strategy of translation is employed by naturally uncapped picornaviral genomic RNAs and several cellular mRNAs. A common feature of these RNAs is a relatively long 5' noncoding region (NCR) that folds into a complex secondary structure harboring an internal ribosome entry site (IRES). Evidence derived from the use of dicistronic expression systems, combined with an extensive mutational analysis, demonstrated the presence of an IRES within the HCV 5'NCR. The results of our continued mutational analysis to map the critical structural elements of the HCV IRES has led to the identification of a pseudoknot structure upstream of the initiator AUG. The evidence presented in this study is based upon the mutational analysis of the putative pseudoknot structure. This is further substantiated by biochemical and enzymatic probing of the wild-type and mutant 5'NCR. Further, the thermodynamic calculations, based upon a modified RNAKNOT program, are consistent with the presence of a pseudoknot structure located upstream of the initiator AUG. Maintenance of this structural element is critical for internal initiation of translation. The pseudoknot structure in the 5'NCR represents a highly conserved feature of all HCV subtypes and members of the pestivirus family, including hog cholera virus and bovine viral diarrhea virus.     

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.     

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.     

A peptide derived from RNA recognition motif 2 of human la protein binds to hepatitis C virus internal ribosome entry site, prevents ribosomal assembly, and inhibits internal initiation of translation

Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.     

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

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

The internal ribosome entry site (IRES) of hepatitis C virus visualized by electron microscopy

Translation of hepatitis C virus (HCV) RNA is initiated via the internal ribosome entry site (IRES), located within the 5' untranslated region. Although the secondary structure of this element has been predicted, little information on the tertiary structure is available. Here we report the first structural characterization of the HCV IRES using electron microscopy. In vitro transcribed RNA appeared as particles with characteristic morphology and gold labeling using a specific oligonucleotide confirmed them to be HCV IRES. Dimerization of the IRES by hybridization with tandem repeat oligonucleotides allowed the identification of domain III and an assignment of domains II and IV to distinct regions within the molecule. Using immunogold labeling, the pyrimidine tract binding protein (PTB) was shown to bind to domain III. Structure-function relationships based on the flexible hinge between domains II and III are suggested. Finally, the architecture of the HCV IRES was seen to be markedly different from that of a picornavirus, foot-and-mouth disease virus (FMDV).     

Duck Hepatitis A virus possesses a distinct type IV internal ribosome entry site element of picornavirus

Sequence analysis of duck hepatitis virus type 1 (DHV-1) led to its classification as the only member of a new genus, Avihepatovirus, of the family Picornaviridae, and so was renamed duck hepatitis A virus (DHAV). The 5' untranslated region (5' UTR) plays an important role in translation initiation and RNA synthesis of the picornavirus. Here, we provide evidence that the 651-nucleotide (nt)-long 5' UTR of DHAV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and within BHK cells. Comparative sequence analysis showed that the 3' part of the DHAV 5' UTR is similar to the porcine teschovirus 1 (PTV-1) IRES in sequence and predicted secondary structure. Further mutational analyses of the predicted domain IIId, domain IIIe, and pseudoknot structure at the 3' end of the DHAV IRES support our predicted secondary structure. However, unlike the case for the PTV-1 IRES element, analysis of various deletion mutants demonstrated that the optimally functional DHAV IRES element with a size of approximately 420 nt is larger than that of PTV-1 and contains other peripheral domains (Id and Ie) that do not exist within the type IV IRES elements. The domain Ie, however, could be removed without significant loss of activity. Surprisingly, like the hepatitis A virus (HAV) IRES element, the activity of DHAV IRES could be eliminated by expression of enterovirus 2A protease. These findings indicate that the DHAV IRES shares common features with type IV picornavirus IRES elements, whereas it exhibits significant differences from type IV IRESs. Therefore, we propose that DHAV possesses a distinct type IV IRES element of picornavirus.     

Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors

Translation initiation of hepatitis C virus (HCV) occurs through an internal ribosome entry site (IRES) located at its 5'-end. As a positive-stranded RNA virus, HCV uses its genome as a common template for translation and replication, but the coordination between these two processes remains poorly characterized. Moreover, although genetic evidence of RNA-protein interactions for viral replication is accumulating because of subgenomic replicons and a recent culture system for HCV, such interactions are still contentious in the regulation of translation. To gain insight into such mechanisms, we addressed the involvement of cis and trans viral factors in HCV IRES activity by using a cell-based RNA reporter system. We found that the HCV 3' noncoding region (NCR) strongly stimulates IRES efficiency in cis, depending on the genotype and the cell line. Moreover, we confirmed the role of the core protein in viral gene expression as previously reported in vitro. Surprisingly, we observed a similar effect, i.e. a twofold increase under low amounts of NS5B RNA polymerase, followed by a decrease at higher concentrations. However, no contribution of NS5A to HCV IRES-mediated translation was noted and no cooperative effect could be detected between 3' NCR and viral proteins or between proteins. Collectively, these results suggest that HCV RNA translation is regulated, and that the switch from translation to replication might involve a sequential requirement for both cis and trans viral factors, because of their apparent lack of synergy, probably with the aid of host factors.     

Cell proteins bind to a linear polypyrimidine-rich sequence within the 5'-untranslated region of rhinovirus 14 RNA.

Members of the picornavirus family initiate translation of their RNA genomes by a cap-independent mechanism in which ribosomes bind to an internal site in the 5' untranslated region (5'-UTR). This unique process requires an internal ribosome entry site (IRES), a highly structured RNA whose function is mediated in part by interactions with cell proteins. The IRES element of human rhinovirus 2 (HRV-2) extends from nucleotide (nt) 10 to between nt 544 and 568 and has been shown to interact with two cell proteins, pyrimidine tract-binding protein (pPTB) and p97. To map the specific regions of HRV-14 RNA that bind cell proteins, mobility shift, UV cross-linking and Western immunoblot analyses were performed. The results indicate that an RNA sequence from nt 538 to 591 interacts with pPTB and La, two proteins previously shown to functionally interact with the IRES elements of several picornaviruses. Two additional proteins, p97 and p68, were also cross-linked to nt 538 to 591 RNA. These four proteins interact with a putatively unstructured portion of the 5'-UTR that contains a polypyrimidine tract and has been shown to be present at the 3' border of sequences that are essential for IRES function of HRV-2. These protein-RNA interactions are likely to play a role in internal initiation of translation.     

trans complementation by RNA of defective foot-and-mouth disease virus internal ribosome entry site elements.

A region of about 435 bases from the 5' noncoding region of foot-and-mouth disease virus RNA directs internal initiation of protein synthesis. This region, termed the internal ribosome entry site (IRES), is predicted to contain extensive secondary structure. Precise deletion of five predicted secondary structure features has been performed. The mutant IRES elements have been constructed into vectors which express bicistronic mRNAs and assayed within cells. Each of the modified IRES elements was defective in directing internal initiation when assayed alone. However, coexpression of an intact foot-and-mouth disease virus IRES complemented four of these defective elements to an efficiency of up to 80% of wild-type activity. No complementation was observed with the structurally analogous element from encephalomyocarditis virus. The role of RNA-RNA interactions in the function of the picornavirus IRES is discussed.     

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for replication of this virus.     

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.