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Internal Ribosome Entry Site (internal + ribosome_entry_site)
Selected AbstractsA picture says more than a thousand words: Structural insights into hepatitis C virus translation initiation,HEPATOLOGY, Issue 6 2006Pantxika Bellecave Ph.D. Protein synthesis in mammalian cells requires initiation factor eIF3, a ,750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5,-cap or an internal ribosome entry site (IRES). Cryoelectron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5,-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly. [source] Immune enhancement of nitroreductase-induced cytotoxicity: Studies using a bicistronic adenovirus vectorINTERNATIONAL JOURNAL OF CANCER, Issue 1 2003Nicola K. Green Abstract The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in preclinical studies and is currently being assessed in phase I clinical trials. It is well established that there is an immune component to the bystander effect observed with other systems such as thymidine kinase and cytosine deaminase; however, such an effect has not previously been described using NR. We have preliminary data suggesting an immune bystander effect with NR to further examine these effects and their potential enhancement by cytokines, an adenoviral vector containing CMV-NR, an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF) was constructed. The NR-GM-CSF virus was validated in 2 experimental models and demonstrated increased therapeutic efficacy in the MC26 murine colorectal tumour model. These data illustrate that the combination of suicide gene therapy using NR and CB1954 with immune stimulation via GM-CSF gives an improved response compared to either modality alone and suggests that the immune component of this response may be beneficial in combating unresectable, metastatic disease and preventing tumour recurrence. © 2002 Wiley-Liss, Inc. [source] Cell specific internal translation efficiency of Epstein,Barr virus present in solid organ transplant patientsJOURNAL OF MEDICAL VIROLOGY, Issue 5 2007Åsa Isaksson Abstract The U leader exon in the 5, untranslated region of the Epstein,Barr virus nuclear antigen 1 (EBNA1) gene contains an internal ribosome entry site, the EBNA internal ribosome entry segment (IRES), which promotes cap-independent translation and increases the expression level of the EBNA1 protein. It was previously reported that immunosuppressed organ transplanted patients showed an alternatively spliced EBNA1 transcript, excluding the EBNA IRES element. To further investigate the function of the EBNA IRES, sequence analysis of the EBNA IRES mRNA was performed in samples from seven organ transplant patients. Two nucleotide changes, G,,,A at position 67531 and C,,,U at position 67585 were found in the EBNA IRES mRNA, relative to the corresponding genomic Epstein,Barr virus (EBV) sequence in all patients. Moreover, the patient derived EBNA IRES mRNA was shown to differ from the IRES mRNA derived from the cell line B95.8 at position 67531 and from the cell lines Rael and P3HR1 at positions 67531 and 67585. cDNA from the various EBNA IRES sequences were cloned into bicistronic vectors, respectively, and used in transient transfection experiments in six human cell lines. The patient specific sequence significantly decreased the IRES activity in T-cells, while the base changes had no significant impact on the activity in B- or in epithelial cells. The genetic mechanisms behind EBV-associated diseases are complex, involving gene regulation by alternative promoters, alternative splicing, and translational control. The nucleotide changes in the patient specific EBNA IRES transcript and its influence on the translational activity, might illustrate new strategies utilised by the EBV to adapt to the immune control in patients with EBV associated diseases. J. Med. Virol. 79:573,581, 2007. © 2007 Wiley-Liss, Inc. [source] Possible involvement in oncogenesis of a single base mutation in an internal ribosome entry site of Epstein,Barr nuclear antigen 1 mRNAJOURNAL OF MEDICAL VIROLOGY, Issue 4 2004Rika Endo Abstract It has been reported recently that the U leader exon located within the 5, untranslated region of Epstein,Barr nuclear antigen 1 (EBNA1) gene contains an internal ribosome entry site (IRES) element. Sequence analysis of the U leader exon was undertaken in samples from 19 patients with infectious mononucleosis and 19 patients with lethal lymphoproliferative diseases and in 15 spontaneously established lymphoblastoid cell lines. The sequence was conserved except for a single base substitution (T-C) at position 67,585. Although the mutation was detected in only one case of infectious mononucleosis, it was found in more than half of the lethal lymphoproliferative diseases and all lymphoblastoid cell lines. The results suggest that a mutation in the IRES element affects EBNA1 gene expression at the translational level and provides Epstein,Barr virus (EBV)-infected cells with a growth advantage, leading to immortalization of cells in vitro and to the development of lethal lymphoproliferative diseases in vivo. J. Med. Virol. 72:630,634, 2004. © 2004 Wiley-Liss, Inc. [source] A novel inducible tyrosine kinase receptor to regulate signal transduction and neurite outgrowthJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2009Ronald W. Alfa Abstract Nervous system growth factor gene delivery can promote axonal growth and prevent cell death in animal models of CNS trauma and neurodegenerative diseases. The ability to regulate growth factor expression or signaling pathways downstream from growth factor receptors remains a desirable goal for in vivo gene transfer. To achieve precise pharmacological modulation of neurotrophin activity, we have generated a chimeric trkA receptor (ItrkA) by fusing the entire intracellular domain of the trkA high-affinity NGF receptor to two intracellular, modified FK506 binding domains for the synthetic small molecule dimerization ligand AP20187. Rat PC12 cells were transduced with lentiviral vectors containing ItrkA and green fluorescent protein (GFP; via an internal ribosome entry site). Treatment of ItrkA-expressing PC12 cells with AP20187 induced neurite outgrowth and differentiation in a time- and dose-dependent fashion, with a half-maximal response at a concentration of 1 nM AP20187. Seventy percent of cells responded to AP20187 by day 3. Western blots demonstrated that AP20187 treatment resulted in phosphorylation of Erk1/2 and Akt in ItrkA-transduced PC12 cells but not in nontransduced, naïve cells. Phosphorylation levels were comparable to levels obtained with 50 ng/ml nerve growth factor (NGF). In addition, ItrkA lentiviral transduction of primary E15 dorsal root ganglion neurons significantly increased neurite growth three- to fourfold in the presence of AP20187 compared with control GFP transduced and naïve neurons. These results demonstrate that small ligand-induced dimerization of the intracellular domain of trkA can efficiently simulate the biological activity of NGF and provide a means to regulate intracellular neurotrophin receptor signaling. © 2009 Wiley-Liss, Inc. [source] Normal nigrostriatal innervation but dopamine dysfunction in mice carrying hypomorphic tyrosine hydroxylase allelesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2003Susanna Althini Abstract We investigated the use of the mouse tyrosine hydroxylase (TH) gene to drive knock-in constructs in catecholaminergic neurons. Two targeting constructs representing truncated forms of either of the BMP receptors ALK-2 or BMPR-II preceded by an internal ribosome entry site (IRES) were introduced into the 3, untranslated region of TH. An frt-flanked neomycin-resistance (neor) cassette was placed in the 3, end of the targeting constructs. Mice homozygous for the knock-in alleles showed various degrees of hypokinetic behavior, depending mainly on whether the neor cassette was removed. In situ hybridization and immunohistochemistry showed that TH mRNA and protein were variously down-regulated in these mouse strains. Reduced levels of dopamine and noradrenalin were found in several brain areas. However, number and morphology of neurons in substantia nigra and their projections to striatum appeared normal in the neor -positive TH hypomorphic mice as examined by markers for L-aromatic amino acid decarboxylase and the dopamine transporter. Elimination of the neor cassette from the knock-in alleles partially restored TH and dopamine levels. The present neor -positive TH hypomorphic mice show that nigrostriatal innervation develops independently of TH and should find use as a model for conditions of reduced catecholamine synthesis, as seen in, for example, L-dihydroxyphenylalanine-responsive dystonia/infantile parkinsonism. © 2003 Wiley-Liss, Inc. [source] Effects of adenoviral-mediated coexpression of bone morphogenetic protein-7 and insulin-like growth factor-1 on human periodontal ligament cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2010L. Yang Yang L, Zhang Y, Dong R, Peng L, Liu X, Wang Y, Cheng X. Effects of adenoviral-mediated coexpression of bone morphogenetic protein-7 and insulin-like growth factor-1 on human periodontal ligament cells. J Periodont Res 2010; 45: 532,540. © 2010 John Wiley & Sons A/S Background and Objective:, Bone morphogenetic protein-7 (BMP-7) and insulin-like growth factor-1 (IGF-1) are important in periodontal reconstruction. However, their synergistic effect in periodontal regeneration by gene delivery has not been reported. In this study, gene delivery of these two growth factors to human periodontal ligament cells (hPDLCs) was examined for its effects on cell proliferation and differentiation. Material and Methods:, Recombinant adenoviruses containing both human BMP-7 and IGF-1 cDNA created by introducing the internal ribosome entry site (IRES) sequence were used to transfer the genes into hPDLCs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis were used to observe their effects on cell proliferation, while alkaline phosphatase activity measurement, RT-PCR and in vivo tests were conducted to investigate their effects on cell differentiation. Results:, The proliferation of hPDLCs transduced by adenoviruses coexpressing BMP-7 and IGF-1 was suppressed while their differentiation ability was enhanced. There was a synergism of BMP-7 and IGF-1 in up-regulating alkaline phosphatase activity and mRNA levels of collagen type I and Runx2. Implantation in vivo with scaffolds illustrated that the transduced cells exhibited osteogenic differentiation and formed bone-like structures. Conclusion:, The combined delivery of BMP-7 and IGF-1 genes using an IRES-based strategy synergistically enhanced differentiation of hPDLCs. It is suggested that this could be a new potential method in gene therapy for periodontal reconstruction. [source] Hepatitis A virus (HAV) proteinase 3C inhibits HAV IRES-dependent translation and cleaves the polypyrimidine tract-binding proteinJOURNAL OF VIRAL HEPATITIS, Issue 9 2010T. Kanda Summary., Hepatitis A virus (HAV) infection is still an important issue worldwide. A distinct set of viruses encode proteins that enhance viral cap-independent translation initiation driven by an internal ribosome entry site (IRES) and suppress cap-dependent host translation. Unlike cytolytic picornaviruses, replication of HAV does not cause host cell shut off, and it has been questioned whether HAV proteins interfere with its own and/or host translation. HAV proteins were coexpressed in Huh-7 cells with reporter genes whose translation was initiated by either cap-dependent or cap-independent mechanisms. Among the proteins tested, HAV proteinase 3C suppressed viral IRES-dependent translation. Furthermore, 3C cleaved the polypyrimidine tract-binding protein (PTB) whose interaction with the HAV IRES had been demonstrated previously. The combined results suggest that 3C-mediated cleavage of PTB might be involved in down-regulation of viral translation to give way to subsequent viral genome replication. [source] Correlation between translation efficiency and outcome of combination therapy in chronic hepatitis C genotype 3JOURNAL OF VIRAL HEPATITIS, Issue 2 2006A. Yasmeen Summary., Combination therapy with interferon- , (IFN- ,) and ribavirin (RBV) in chronic hepatitis C demonstrates the best responses against hepatitis C virus (HCV) of genotype 3. Still, it has proven to be ineffective in 20,30% of patients infected with this genotype. In the present study, we analysed the translation efficiency mediated by the internal ribosome entry site (IRES) region in HCV genotype 3 genomes isolated from sustained responders (SR) and non-responders (NR), assuming that this may influence the outcome of treatment. Pretreatment isolates of genotype 3 from 22 individuals (15 SR, seven NR) were selected for such analyses. The IRES region [nucleotide (nt) 1,407] was cloned into a dual luciferase vector and IRES activity assessed following transfection into various cell lines. Low relative translation efficiency was observed for IRES elements derived from SR patients, whereas those of NR patients showed significantly greater translation efficiency (29.7 ± 13 vs 69.4 ± 22; P < 0.01). Subsequently, the effect of IFN- , plus RBV on IRES-driven translation in vitro was determined. A greater suppressive effect was observed on IRES activity isolated from seven SR patients, when compared with seven NR patients. In conclusion, IRES efficiency in vitro correlated with treatment response for HCV genotype 3. Further studies are warranted to investigate whether IRES efficiency in vitro, or sequence motifs associated with IRES efficiency, will be worthwhile to explore as prognostic tools for other HCV genotypes in the treatment of chronic HCV infection. [source] Single nucleotide insertion in the 5,-untranslated region of hepatitis C virus with clearance of the viral RNA in a liver transplant recipient during acute hepatitis B virus superinfectionLIVER INTERNATIONAL, Issue 1 2002Consolato Sergi Abstract: Hepatitis C virus (HCV) infection is an important etiology in patients undergoing orthotopic liver transplantation (OLT) world-wide. Antiviral therapy-related clearance of HCV RNA may occur both in patients with chronic HCV infection and in transplanted patients for HCV-related liver cirrhosis, but the role of the 5,-untranslated region (UTR) of HCV containing the internal ribosome entry site (IRES), which directs the translation of the viral open reading frame has not hitherto been evaluated. We studied the 5,-UTR in an HCV-infected recipient of a liver graft that showed spontaneous clearance of HCV RNA during an acute hepatitis B virus (HBV) superinfection. Sequencing of the 5,-UTR of HCV showed a nucleotide A insertion at position 193 of the IRES. [source] Implementation in Australia of molecular diagnostic techniques for the rapid detection of foot and mouth disease virusAUSTRALIAN VETERINARY JOURNAL, Issue 7 2004DB BOYLE Objective To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia. Design Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks. Results Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3. Conclusion Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia. [source] A novel synthetic mammalian promoter derived from an internal ribosome entry siteBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2006Shizuka Hartenbach Abstract Introduction of specific mutations into a synthetic internal ribosome entry site (IRESGTX) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic PGTX promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high-level expression of a variety of transgenes including the human vascular endothelial growth factor 121 (VEGF121), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus -derived secreted ,-amylase (SAMY) in Chinese hamster ovary cells (CHO-K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleotide (resulting in PGTX2) doubled the SEAP expression levels in CHO-K1. PGTX2 reached near 70% of PSV40 -driven expression levels and outperformed constitutive phosphoglycerate kinase (PPGK) and human ubiquitin C (PhUBC) promoters in CHO-K1. Also, PGTX2 was successfully engineered for macrolide-inducible transgene expression. Owing to its size of only 182 bp, PGTX2 is one of the smallest eukaryotic promoters. Although PGTX2 was found to be a potent promoter, it retained its IRESGTX -specific translation-initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells. © 2006 Wiley Periodicals, Inc. [source] | |||||||||||||