Internal Ribosome Entry Site Function Research Articles

IL-6 Enhances C-myc Translation in Myeloma Cells Via Activation of the Myc Internal Ribosomal Entry Site (IRES) and an IRES-Binding Trans Factor, hnRNP A1.

Dysregulated c-myc expression in multiple myeloma (MM) can be due to a translocation with juxtaposition of the myc gene to the Ig enhancer resulting in constitutive transcription. However, myc protein levels can also be increased in MM cells when it is due to heightened translation. Activated translation can be mediated via an mTOR-mediated, cap-dependent mechanism, thus named because the translation initiation complex utilizes the cap structure on mRNA to recruit the ribosomal subunit. However, an alternative mechanism is cap-independent where the ribosomal subunit binds a transcript via an IRES in the RNA's 5′UTR. IRES function is regulated by IRES trans activating factors (ITAFs) which bind to the 5′UTR. We studied myc expression in ANBL-6 wild type and activated K-RAS-transfected MM cells which proliferatively respond to IL-6 but do not contain myc translocations. IL-6 increased myc protein expression in both cell lines in a concentration and time-dependent fashion but RNA expression was only enhanced in the wild type cells. The increased protein expression in K-RAS mutant cells was unaffected by rapamycin, indicating independence from mTOR and cap-dependent mechanisms. Using an IRES-dependent reporter vector, reporter expression was shown to be increased by IL-6, confirming IL-6 activates myc IRES activity. This was also demonstrated in the IL-6 autocrine U266 MM cell line where myc IRES-dependent reporter expression was ablated following exposure to anti-IL-6 antibodies. In addition, primary MM cells treated with IL-6 also demonstrated an increase in myc IRES activity. Since a yeast three-hybrid screen for proteins specifically binding to the myc IRES identified several clones encoding hnRNP A1, we evaluated hnRNP A1 as a mediator of myc IRES activity. IL-6 increased the cytoplasmic localization of hnRNP A1 in ANBL-6 MM cells. Furthermore, in MM cells where hnRNP A1 expression was knocked down, myc IRES activity was greatly diminished and there was no enhancement with addition of IL-6. These results indicate that IL-6 can enhance c-myc translation in MM cells via activation of the myc IRES and that hnRNP A1 is a critical myc ITAF involved in the IL-6 response. hnRNP A1 may serve as a new therapeutic target in myeloma.

The Role of AKT and ERK in Regulating D-Cyclin Translation Inhibition and G1 Arrest in Multiple Myeloma Cells Treated with mTOR Inhibitors: Rationale for Combination Thereapy.

In a previous study, we showed that heightened AKT activity sensitized multiple myeloma (MM) cells to the in vivo anti-tumor effects of CCI-779. To test the mechanism of AKT's regulatory role, we studied isogenic U266 MM cell lines transfected with an activated AKT allele or empty vector. The AKT-transfected cells were markedly more sensitive to cytostasis induced in vitro by rapamycin or in vivo by CCI-779. In contrast, cells with quiescent AKT were completely resistant. The ability of rapamycin and CCI-779 to inhibit D-cyclin expression was also significantly greater in AKT-transfected MM cells and this was, in part, due to a greater ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. As ERK/p38 activity can facilitate IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of rapamycin sensitivity. AKT-transfected cells demonstrated significantly decreased ERK and p38 activity, suggesting their involvement. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells while a p38 inhibitor had no effect. Furthermore, the combination of rapamycin and the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. These data support a scenario where ERK facilitates D-cyclin IRES function and heightened AKT activity down regulates this ERK-dependent phenomenon. Thus ERK and AKT activity are potential predictors of responsiveness to mTOR inhibitors.

In vivo footprint of a picornavirus internal ribosome entry site reveals differences in accessibility to specific RNA structural elements

Internal ribosome entry site (IRES) elements were described in picornaviruses as an essential region of the viral RNA. Understanding of IRES function requires a detailed knowledge of each step involved in the internal initiation process, from RNA folding and IRES-protein interaction to ribosome recruitment. Thus, deciphering IRES accessibility to external agents due to RNA structural features, as well as RNA-protein protection within living cells, is of primary importance. In this study, two chemical reagents, dimethylsulfate (DMS) and aminomethylpsoralen, have been used to footprint the entire IRES of foot-and-mouth disease virus (FMDV) in living cells; these reagents enter the cell membrane and interact with nucleic acids in a structure-dependent manner. For FMDV, as in other picornaviruses, viral infection is dependent on the correct function of the IRES; therefore, the IRES region itself constitutes a useful target of antiviral drugs. Here, the in vivo footprint of a picornavirus IRES element in the context of a biologically active mRNA is shown for the first time. The accessibility of unpaired adenosine and cytosine nucleotides in the entire FMDV IRES was first obtained in vitro by DMS probing; subsequently, this information was used to interpret the footprint data obtained in vivo for the mRNA encompassing the IRES element in the intercistronic space. The results of DMS accessibility and UV-psoralen cross-linking studies in the competitive cellular environment provided evidence for differences in RNA structure from data obtained in vitro, and provided essential information to identify appropriate targets within the FMDV IRES aimed at combating this important pathogen.

Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain.

Hepatitis C virus (HCV) translation initiation depends on an internal ribosome entry site (IRES). We previously identified an RNA molecule (HH363-10) able to bind and cleave the HCV IRES region. This paper characterizes its capacity to interfere with IRES function. Inhibition assays showed that it blocks IRES activity both in vitro and in a human hepatoma cell line. Although nucleotides involved in binding and cleavage reside in separate regions of the inhibitor HH363-10, further analysis demonstrated the strongest effect to be an intrinsic feature of the entire molecule; the abolishment of either of the two activities resulted in a reduction in its function. Probing assays demonstrate that HH363-10 specifically interacts with the conserved IIIf domain of the pseudoknot structure in the IRES, leading to the inhibition of the formation of translationally competent 80S particles. The combination of two inhibitory activities targeting different sequences in a chimeric molecule may be a good strategy to avoid the emergence of resistant viral variants.

Ectropis obliqua picorna-like virus IRES-driven internal initiation of translation in cell systems derived from different origins

Ectropis obliqua picorna-like virus (EoPV) is an insect RNA virus that causes a lethal granulosis infection of larvae of the tea looper (Ectropis obliqua). An internal ribosome entry site (IRES) mediates translation initiation of EoPV RNA. Here, bicistronic constructs were used to examine the 5' untranslated region (UTR) of EoPV for IRES activity. The capacities of the EoPV 5' UTR IRES and another insect virus IRES, the cricket paralysis virus intergenic region IRES, to mediate internal translation initiation in a variety of translation systems were also compared. The results demonstrated that the EoPV IRES functioned efficiently not only in mammalian cell-derived systems, but also in an insect cell-derived translation system. However, it functioned inefficiently in a plant cell-derived translation system. This study reveals the host preferences of the EoPV IRES and important differences in IRES function between the EoPV IRES and other characterized picorna-like insect viral IRESs.

A Ferritin-responsive Internal Ribosome Entry Site Regulates Folate Metabolism

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.

Conservation and Diversity Among the Three-dimensional Folds of the Dicistroviridae Intergenic Region IRESes

Internal ribosome entry site (IRES) RNAs are necessary for successful infection of many pathogenic viruses, but the details of the RNA structure-based mechanism used to bind and manipulate the ribosome remain poorly understood. The IRES RNAs from the Dicistroviridae intergenic region (IGR) are an excellent model system to understand the fundamental tenets of IRES function, requiring no protein factors to manipulate the ribosome and initiate translation. Here, we explore the architecture of four members of the IGR IRESes, representative of the two divergent classes of these IRES RNAs. Using biochemical and structural probing methods, we show that despite sequence variability they contain a common three-dimensional fold. The three-dimensional architecture of the ribosome binding domain from these IRESes is organized around a core helical scaffold, around which the rest of the RNA molecule folds. However, subtle variation in the folds of these IRESes and the presence of an additional secondary structure element suggest differences in the details of their manipulation of the large ribosomal subunit. Overall, the results demonstrate how a conserved three-dimensional RNA fold governs ribosome binding and manipulation.

Identification of minimal sequences of the Rhopalosiphum padi virus 5′ untranslated region required for internal initiation of protein synthesis in mammalian, plant and insect translation systems

Rhopalosiphum padi virus (RhPV) is a member of the family Dicistroviridae. The genomes of viruses in this family contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The RhPV 5' IRES is functional in mammalian, insect and plant translation systems and can form 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and eIF1. Large regions of the 5' untranslated region (UTR) can be deleted without affecting initiation-complex formation. The minimal sequences required for directing internal initiation in mammalian (rabbit reticulocyte lysate), plant (wheatgerm extract) and insect (Sf21 cells) translation systems have now been defined. A fragment (nt 426-579) from the 3' portion of the 5' UTR can direct translation in each of these translation systems. In addition, a distinct region (nt 300-429) is also active. Thus, unstructured regions within the 5' UTR seem to be critical for IRES function.

Protein Kinase C Regulates Internal Initiation of Translation of the GATA-4 mRNA following Vasopressin-induced Hypertrophy of Cardiac Myocytes

GATA-4 is a key member of the GATA family of transcription factors involved in cardiac development and growth as well as in cardiac hypertrophy and heart failure. Our previous studies suggest that GATA-4 protein synthesis may be translationally regulated. We report here that the 518-nt long 5'-untranslated region (5'-UTR) of the GATA-4 mRNA, which is predicted to form stable secondary structures (-65 kcal/mol) such as to be inhibitory to cap-dependent initiation, confers efficient translation to monocistronic reporter mRNAs in cell-free extracts. Moreover, uncapped GATA-4 5'-UTR containing monocistronic reporter mRNAs continue to be well translated while capped reporters are insensitive to the inhibition of initiation by cap-analog, suggesting a cap-independent mechanism of initiation. Utilizing a dicistronic luciferase mRNA reporter containing the GATA-4 5'-UTR within the intercistronic region, we demonstrate that this leader sequence confers functional internal ribosome entry site (IRES) activity. The activity of the GATA-4 IRES is unaffected in trans-differentiating P19CL6 cells, however, is strongly stimulated immediately following arginine-vasopressin exposure of H9c2 ventricular myocytes. IRES activity is then maintained at submaximal levels during hypertrophic growth of these cells. Supraphysiological Ca(2+) levels diminished stimulation of IRES activity immediately following exposure to vasopressin and inhibition of protein kinase C activity utilizing a pseudosubstrate peptide sequence blocked IRES activity during hypertrophy. Thus, our data suggest a mechanism for GATA-4 protein synthesis under conditions of reduced global cap-dependent translation, which is maintained at a submaximal level during hypertrophic growth and point to the regulation of GATA-4 IRES activity by sarco(ER)-reticular Ca(2+) stores and PKC.

Picornavirus Internal Ribosome Entry Site Elements Can Stimulate Translation of Upstream Genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.

Alphavirus replicon approach to promoterless analysis of IRES elements

Here we describe a system for promoterless analysis of putative internal ribosome entry site (IRES) elements using an alphavirus (family Togaviridae) replicon vector. The system uses the alphavirus subgenomic promoter to produce transcripts that, when modified to contain a spacer region upstream of an IRES element, allow analysis of cap-independent translation of genes of interest (GOI). If the IRES element is removed, translation of the subgenomic transcript can be reduced > 95% compared to the same transcript containing a functional IRES element. Alphavirus replicons, used in this manner, offer an alternative to standard dicistronic DNA vectors or in vitro translation systems currently used to analyze putative IRES elements. In addition, protein expression levels varied depending on the spacer element located upstream of each IRES. The ability to modulate the level of expression from alphavirus vectors should extend the utility of these vectors in vaccine development.

In vitro and in vivo identification of structural and sequence elements in the 5′ untranslated region of Ectropis obliqua picorna-like virus required for internal initiation

Ectropis obliqua picorna-like virus (EoPV) is a newly described insect virus that is classified as a putative member of the genus Iflavirus. The virus possesses a large, positive-sense RNA genome encoding a single polyprotein that shares physicochemical properties with those of members of the family Picornaviridae. The 5' untranslated region (5' UTR) plays an important role in picornavirus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation. To investigate translation in EoPV, an extensive range of mutations were engineered within the 5' UTR and the effects of these changes were examined in vitro and in vivo by using a bicistronic construct. Results showed that deletions within the first 63 nt had little impact on IRES activity, whilst core IRES function was contained within stem-loops C and D, as their removal abrogated IRES activity significantly. In contrast to these findings, removal of stem-loop G containing two cryptic AUGs caused a remarkable increase in IRES activity, which was further investigated by site-directed mutagenesis at these two positions. It was also confirmed that initiation of protein synthesis occurs at AUG6 (position 391-394) and not at the AUG immediately downstream of the polypyrimidine tract. Mutation of the polypyrimidine tract (CCTTTC) had a slight effect on EoPV IRES activity. Furthermore, mutations of the RAAA motif led to a decrease in IRES activity of approximately 40 % in vitro, but these results were not supported by in vivo experiments. In conclusion, this study reveals that the EoPV IRES element is unique, although it has features in common with the type II IRESs.

AKT activity regulates the ability of mTOR inhibitors to prevent angiogenesis and VEGF expression in multiple myeloma cells

We recently demonstrated that the mammalian target of rapamycin (mTOR) inhibitor, CCI-779, curtailed the growth of a subcutaneous challenge of multiple myeloma (MM) cells in immunodeficient mice. This antitumor effect was associated with prevention of cell proliferation, induction of apoptosis and inhibition of angiogenesis. Interestingly, myeloma tumors with heightened AKT activation were particularly sensitive to a CCI-779-induced antitumor response. To investigate whether part of the differential sensitivity was due to an AKT-regulated effect on angiogenesis, we compared the effects of mTOR inhibitors against isogenic MM cell lines that only differ by their degree of AKT activity. In this model, heightened AKT activity significantly sensitized MM cells to the following inhibitory effects of mTOR inhibition: angiogenesis in vivo, vascular endothelial growth factor (VEGF) expression in vitro and in vivo and VEGF translation (but not transcription). Assessment of p70S6 kinase activity indicated that rapamycin induced comparable mTOR inhibition in both cell lines suggesting that an adverse effect on VEGF cap-dependent translation would be comparable. Internal ribosome entry site (IRES)-mediated cap-independent translation is a salvage pathway for protein expression when mTOR is inhibited, so we analyzed a possible regulatory role of AKT on VEGF IRES activity. We found that elevated AKT activity inhibited VEGF IRES function. These results support a mechanism whereby AKT prevents VEGF IRES activity in myeloma cells during mTOR inhibition resulting in a more complete abrogation of VEGF translation, and ultimately, angiogenesis.

Caliciviruses Differ in Their Functional Requirements for eIF4F Components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

19S Late mRNAs of Simian Virus 40 Have an Internal Ribosome Entry Site Upstream of the Virion Structural Protein 3 Coding Sequence

The late mRNAs of simian virus 40 (SV40) are polycistronic. The 19S mRNAs encode primarily the virion structural proteins VP2 and VP3. The VP2 and VP3 coding sequences are located in the same reading frame, and the VP3 AUG is an internal AUG for VP2. We tested whether an internal ribosome entry site (IRES) might be located upstream of the VP3 AUG that would facilitate its utilization, especially late in infection when cap-dependent translation is reduced (19). Using dicistronic reporter systems for IRES detection, we detected IRES activity within SV40 nucleotides (nts) 565 to 916, the region between the VP2 and VP3 AUGs. Nuclease protection analysis and primer extension analysis indicate no aberrant transcription or splicing that could account for false prediction of an IRES. Deletion analysis of the region indicates the presence of two functional IRESs, one within nts 661 to 830 and the other within nts 771 to 915. These two regions, each containing one IRES, have essentially the same IRES activity as the entire region spanning nts 616 to 915, which contains both IRESs. This suggests that potential secondary structures in the overlapping regions spanning nts 661 to 830 and nts 771 to 915 may be in dynamic equilibrium, such that there is only one functional IRES at any one time. These data strongly suggest that an IRES can be utilized for VP3 translation from the SV40 19S late mRNAs.

Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by ViralcisandtransFactors

Enteroviruses (EVs) overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap-independent manner at the viral internal ribosomal entry site (IRES). We have investigated the role of cis- and trans-acting viral factors in EV IRES translation in living cells. We observed that considerable portions of the viral genome, including the 5'-proximal open reading frame and the 3' untranslated region, contribute to stimulation of IRES-mediated translation. With the IRES in proper context, translation via internal initiation in uninfected cells is as efficient as at capped messages with short, unstructured 5' untranslated regions. IRES function is enhanced in cells infected with the EV coxsackievirus B3, but the related poliovirus has no significant stimulatory activity. This differential is due to the inherent properties of their 2A protease and is not coupled to 2A-mediated proteolytic degradation of the eukaryotic initiation factor 4G. Our results suggest that the efficiency of alternative translation initiation at EV IRESs depends on a properly configured template rather than on targeted alterations of the host cell translation machinery.

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.

Treatment of CNS Malignancies with a Recombinant Oncolytic Poliovirus

Spread to the central nervous system (CNS) and the leptomeninges is a frequent complication of systemic cancers that is associated with serious morbidity and high mortality. We have developed a novel therapeutic approach against primary CNS tumors and secondary CNS malignancy complicating systemic cancer based on the human neuropathogen poliovirus. Susceptibility to poliovirus infection and ensuing rapid cell lysis are mediated by the cellular poliovirus receptor CD155, as well as cell-internal factors involved in control of viral gene expression and genome replication. We observed that CD155, a putative cell adhesion molecule of the immunoglobulin superfamily physiologically expressed in the developing CNS, is ectopically expressed on numerous primary and secondary CNS neoplasms. An association with malignant cells renders CD155 a molecular target for therapeutic intervention with poliovirus. However, any therapeutic application of poliovirus must contend with its inherent neuropathogenic potential. Poliovirus gene expression is driven by the viral internal ribosomal entry site (IRES), which mediates translation initiation in a 5- end, cap-independent manner. We discovered that IRES function is subject to potent cell type-specific restrictions that can be exploited to selectively drive viral gene expression and propagation in cancerous cells. Polioviruses replicating under control of a heterologous IRES derived from their relative human rhinovirus type 2, displayed rapid growth and cell killing in cancerous cells. However, these recombinants failed to efficiently translate their genome and propagate in cells of neuronal lineage. Consequently, polio-/rhinovirus recombinants are non-pathogenic after intraspinal inoculation into mice transgenic for the poliovirus receptor CD155 and in non-human primates. Oncolytic poliovirus recombinants have shown promise in preclinical investigations against primary tumor explant cultures and in xenograft animal studies and are currently being prepared for phase-I clinical trials against glioblastoma multiforme.

Hepatitis C Virus Core Protein Acts as a trans-Modulating Factor on Internal Translation Initiation of the Viral RNA

Translation initiation of hepatitis C virus (HCV) RNA occurs through an internal ribosome entry site (IRES) located at its 5' end. As a positive-stranded virus, HCV uses the genomic RNA template for translation and replication, but the transition between these two processes remains poorly understood. HCV core protein (HCV-C) has been proposed as a good candidate to modulate such a regulation. However, current data are still the subject of controversy in attributing any potential role in HCV translation to the HCV core protein. Here we demonstrate that HCV-C displays binding activities toward both HCV IRES and the 40 S ribosomal subunit by using centrifugation on sucrose gradients. To gain further insight into these interactions, we investigated the effect of exogenous addition of purified HCV-C on HCV IRES activity by using an in vitro reporter assay. We found that HCV IRES-mediated translation was specifically modulated by HCV-C provided in trans, in a dose-dependent manner, with up to a 5-fold stimulation of the IRES efficiency upon addition of low amounts of HCV-C, followed by a decrease at high doses. Interestingly, mutations within some domains of the IRES as well as the presence of an upstream reporter gene both lead to changes in the expected effects, consistent with the high dependence of HCV IRES function on its overall structure. Collectively, these results indicate that the HCV core protein is involved in a tight modulation of HCV translation initiation, depending on its concentration, and they suggest an important biological role of this protein in viral gene expression.

Translation of Eukaryotic Translation Initiation Factor 4GI (eIF4GI) Proceeds from Multiple mRNAs Containing a Novel Cap-dependent Internal Ribosome Entry Site (IRES) That Is Active during Poliovirus Infection

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential scaffolding protein required to recruit the 43 S complex to the 5'-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5'-UTR per se. However, the 5'-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5' m7guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of cap-dependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.