A host-specific, temperature-sensitive translation defect determines the attenuation phenotype of a human rhinovirus/poliovirus chimera, PV1(RIPO)

By using a rhinosvirus/poliovirus type 1 chimera, PV1(RIPO), with the cognate internal ribosome entry site (IRES) of human rhinovirus type 2 (HRV2), we set out to shed light on the mechanism by which this variant expresses its attenuated phenotype in poliovirus-sensitive, CD155 transgenic (tg) mice and cynomolgus monkeys. Here we report that replication of PV1(RIPO) is restricted not only in human cells of neuronal origin, as was reported previously, but also in cells of murine origin at physiological temperature. This block in replication was enhanced at 39.5°C but, remarkably, it was absent at 33°C. PV1(RIPO) variants that overcame the replication block were derived by serial passage under restrictive conditions in either mouse cells or human neuronal cells. All adapting mutations mapped to the 5'-nontranslated region of PV1(RIPO). Variants selected in mouse cells, but not in human neuronal cells, exhibited increased mouse neurovirulence in vivo. The observed strong mouse-specific defect of PV1(RIPO) at nonpermissive temperature correlated with the translational activity of the HRV2 IRES in this chimeric virus. These unexpected results must be kept in mind when poliovirus variants are tested in CD155 tg mice for their neurovirulent potential, particularly in assays of live attenuated oral poliovirus vaccine lots. Virulence may be masked by adverse species-specific conditions in mouse cells that may not allow accurate prediction of neurovirulence in the human host. Thus, novel poliovirus variants in line for possible development of human vaccines must be tested in nonhuman primates.     

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.     

Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus

Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (-/+) or both (-/-) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr(+/+), unr(+/-), and unr(-/-) cell lines. Translation directed by the HRV IRES was severely impaired in unr(-/-) cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr(-/-) cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.     

Attenuation of herpes simplex virus neurovirulence with picornavirus cis-acting genetic elements

Viral pathogenesis depends on a suitable milieu in target host cells permitting viral gene expression, propagation, and spread. In many instances, viral genomes can be manipulated to select for propagation in certain tissues or cell types. This has been achieved for the neurotropic poliovirus (PV) by exchange of the internal ribosomal entry site (IRES), which is responsible for translation of the uncapped plus-strand RNA genome. The IRES of human rhinovirus type 2 (HRV2) confers neuron-specific replication deficits to PV but has no effect on viral propagation in malignant glioma cells. We report here that placing the critical gamma(1)34.5 virulence genes of herpes simplex virus type 1 (HSV) under translation control of the HRV2 IRES results in neuroattenuation in mice. In contrast, IRES insertion permits HSV propagation in malignant glioma cell lines that do not support replication of HSV recombinants carrying gamma(1)34.5 deletions. Our observations indicate that the conditions for alternative translation initiation at the HRV2 IRES in malignant glioma cells differ from those in normal central nervous system (CNS) cells. Picornavirus regulatory sequences mediating cell type-specific gene expression in the CNS can be utilized to target cancerous cells at the level of translation regulation outside their natural context.     

Age-dependent poliovirus replication in the mouse central nervous system is determined by internal ribosome entry site-mediated translation

Mouse cells are not permissive for the replication of human rhinovirus type 2 (HRV2). To determine the role of the HRV2 internal ribosome entry site (IRES) in determining species specificity, a recombinant poliovirus (P1/HRV2) was constructed by substituting the poliovirus IRES with the IRES from HRV2. This recombinant virus replicated in all human and murine cell lines examined, demonstrating that the HRV2 IRES does not limit viral replication in transformed murine cells. P1/HRV2 replicated in the brain and spinal cord in neonatal but not adult mice transgenic for the poliovirus receptor, CD155. Passage of P1/HRV2 in mice led to selection of a virus that caused paralysis in neonatal mice. To determine the relationship between HRV2 IRES-mediated translation and replication of P1/HRV2 in mice, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The results demonstrate that the HRV2 IRES mediates translation in organs of neonatal but not adult mice. These findings show that HRV2 IRES-mediated translation is a determinant of virus replication in the murine brain and spinal cord and suggest that the IRES determines the species specificity of HRV2 infection.     

The double-stranded RNA binding protein 76:NF45 heterodimer inhibits translation initiation at the rhinovirus type 2 internal ribosome entry site

Poliovirus (PV) plus-strand RNA genomes initiate translation in a cap-independent manner via an internal ribosome entry site (IRES) in their 5' untranslated region. Viral translation is codetermined by cellular IRES trans-acting factors, which can influence viral propagation in a cell-type-specific manner. Engineering of a poliovirus recombinant devoid of neuropathogenic properties but highly lytic in malignant glioma cells was accomplished by exchange of the cognate poliovirus IRES with its counterpart from human rhinovirus type 2 (HRV2), generating PV-RIPO. Neuroblast:glioma heterokaryon analyses revealed that loss of neurovirulence is due to trans-dominant repression of PV-RIPO propagation in neuronal cells. The double-stranded RNA binding protein 76 (DRBP76) was previously identified to bind to the HRV2 IRES in neuronal cells and to inhibit PV-RIPO translation and propagation (M. Merrill, E. Dobrikova, and M. Gromeier, J. Virol. 80:3347-3356, 2006). The results of size exclusion chromatography indicate that DRBP76 heterodimerizes with nuclear factor of activated T cells, 45 kDa (NF45), in neuronal but not in glioma cells. The DRBP76:NF45 heterodimer binds to the HRV2 IRES in neuronal but not in glioma cells. Ribosomal profile analyses show that the heterodimer preferentially associates with the translation apparatus in neuronal cells and arrests translation at the HRV2 IRES, preventing PV-RIPO RNA assembly into polysomes. Results of this study suggest that the DRBP76:NF45 heterodimer selectively blocks HRV2 IRES-driven translation initiation in neuron-derived cells.     

La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro

Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5' untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.     

Translation of polioviral mRNA is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3C(pro)

The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3C(pro) (and/or 3CD(pro)), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3C(pro) target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA.     

Polypyrimidine-tract binding protein (PTB) is necessary, but not sufficient, for efficient internal initiation of translation of human rhinovirus-2 RNA

Initiation of translation of the animal picornavirus RNAs is via a mechanism of direct internal ribosome entry, which requires a substantial segment of the viral 5'-untranslated region, generally known as the IRES (for "internal ribosome entry site"). Because, however, translation of the RNAs of members of the enterovirus, and more especially, the rhinovirus subgroups of the Picornaviridae is restricted in the reticulocyte lysate system, but is greatly stimulated by the addition of HeLa cell extracts, the implication is that, in these cases, internal initiation also requires cellular trans-acting factors that are more abundant in HeLa cell extracts than in rabbit reticulocytes. This was used as the basis of a functional assay for the purification of the HeLa cell factors required for translation dependent on the human rhinovirus-2 (HRV) IRES. There are two such HeLa cell factors separable by ion-exchange chromatography, each of which is individually active in the assay, although their combined effect is synergistic. One of these activities is shown to be polypyrimidine-tract binding protein (PTB) on the grounds that (1) the activity copurifies to homogeneity with PTB and (2) recombinant PTB expressed in Escherichia coli stimulates HRV IRES-dependent translation with a specific activity similar to that of the purified HeLa cell factor. Furthermore, it is shown that recombinant PTB also stimulates the translation of RNAs bearing the poliovirus type 1 (Mahoney) IRES.     

Molecular mechanisms of attenuation of the Sabin strain of poliovirus type 3

Mutations critical for the central nervous system (CNS) attenuation of the Sabin vaccine strains of poliovirus (PV) are located within the viral internal ribosome entry site (IRES). We examined the interaction of the IRESs of PV type 3 (PV3) and Sabin type 3 (Sabin3) with polypyrimidine tract-binding protein (PTB) and a neural cell-specific homologue, nPTB. PTB and nPTB were found to bind to a site directly adjacent to the attenuating mutation, and binding at this site was less efficient on the Sabin3 IRES than on the PV3 IRES. Translation mediated by the PV3 and Sabin3 IRESs in neurons of the chicken embryo spinal cord demonstrated a translation deficit for the Sabin3 IRES that could be rescued by increasing PTB expression in the CNS. These data suggest that the low levels of PTB available in the CNS, coupled to a reduced binding of PTB on the Sabin3 IRES, leads to its CNS-specific attenuation. This study also demonstrates the use of the chicken embryo to easily investigate translation of RNA within a neuron in the CNS of an intact living organism.     

The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated

A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.     

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.     

HuR and Ago2 Bind the Internal Ribosome Entry Site of Enterovirus 71 and Promote Virus Translation and Replication

EV71 (enterovirus 71) RNA contains an internal ribosomal entry site (IRES) that directs cap-independent initiation of translation. IRES-dependent translation requires the host’s translation initiation factors and IRES-associated trans-acting factors (ITAFs). We reported recently that mRNA decay factor AUF1 is a negative-acting ITAF that binds IRES stem-loop II. We also reported that the small RNA-processing enzyme Dicer produces at least four small RNAs (vsRNAs) from the EV71 IRES. One of these, vsRNA1, derived from IRES stem-loop II, reduces IRES activity and virus replication. Since its mechanism of action is unknown, we hypothesized that it might control association of ITAFs with the IRES. Here, we identified the mRNA stability factor HuR and the RISC subunit Argonaute 2 (Ago2) as two ITAFs that bind stem-loop II. In contrast to AUF1, HuR and Ago2 promote EV71 IRES activity and virus replication. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with stem-loop II. This presents a possible mechanism by which vsRNA1 could control viral translation and replication.     

Dual Stem Loops within the Poliovirus Internal Ribosomal Entry Site Control Neurovirulence

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5′ nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into Cynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.     

Cell-type-specific repression of internal ribosome entry site activity by double-stranded RNA-binding protein 76

Translation of picornavirus plus-strand RNA genomes occurs via internal ribosomal entry at highly structured 5' untranslated regions. In addition to canonical translation factors, translation rate is likely influenced by supplementary host and viral trans-acting factors. We previously reported that insertion of a heterologous human rhinovirus type 2 internal ribosomal entry site (IRES) into the poliovirus (PV) genome, generating the chimeric virus PV-RIPO, selectively abrogates viral translation and propagation in neurons, which eliminate poliovirus's signature neuropathogenicity. While severely deficient in cells of neuronal lineage, the rhinovirus IRES promotes efficient propagation of PV-RIPO in cancer cells. Tumor-specific IRES function can be therapeutically exploited to direct viral cytotoxicity to cancer cells. Neuron-glioma heterokaryon analysis implicates neuronal trans-dominant inhibition in this effect, suggesting that host trans-acting factors repress IRES function in a cell-type-specific manner. We identified a set of proteins from neuronal cells with affinity for the rhinovirus IRES, including double-stranded RNA-binding protein 76 (DRBP76). DRBP76 associates with the IRES in neuronal but not in malignant glioma cells. Moreover, DRBP76 depletion in neuronal cells enhances rhinovirus IRES-driven translation and virus propagation. Our observations suggest that cell-type-specific association of DRBP76 with the rhinovirus IRES represses PV-RIPO translation and propagation in neuronal cells.     

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.     

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.     

Polypyrimidine tract-binding protein inhibits translation of bip mRNA

Translation initiation of human Bip mRNA is directed by an internal ribosomal entry site (IRES) located in the 5' non-translated region. No trans-acting factor possibly involved in this process has as of yet been identified. For the encephalomyocarditis virus and other picornaviruses, polypyrimidine tract-binding protein (PTB) has been found to enhance the translation through IRES elements, probably by interaction with the IRES structure. Here, we report that PTB specifically binds to the central region (nt 50-117) of the Bip 5' non-translated region. Addition of purified PTB to rabbit reticulocyte lysate and overexpression of PTB in Cos-7 cells selectively inhibited Bip IRES-dependent translation. On the other hand, depletion of endogenous PTB or addition of an RNA interacting with PTB enhanced the translational initiation directed by Bip IRES. These suggest that PTB can either enhance or inhibit IRES-dependent translation depending on mRNAs.     

Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.     

A Small Yeast RNA Blocks Hepatitis C Virus Internal Ribosome Entry Site (HCV IRES)-Mediated Translation and Inhibits Replication of a Chimeric Poliovirus under Translational Control of the HCV IRES Element

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma. We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation. Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro. A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized. Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell lines. Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES. In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line. Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.