scholarly journals A B12-responsive Internal Ribosome Entry Site (IRES) Element in Human Methionine Synthase

2005 ◽  
Vol 280 (38) ◽  
pp. 32662-32668 ◽  
Author(s):  
Sebastian Oltean ◽  
Ruma Banerjee
Keyword(s):  
Internal Ribosome Entry Site ◽  
Methionine Synthase ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element

scholarly journals A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 42-50 ◽  
Author(s):  
RA Sokolic ◽  
S Sekhsaria ◽  
Y Sugimoto ◽  
N Whiting-Theobald ◽  
GF Linton ◽  
...  
Keyword(s):  
Gene Therapy ◽  
B Cells ◽  
Resistance Gene ◽  
Internal Ribosome Entry Site ◽  
Therapeutic Gene ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element ◽  
O2 Production

Abstract Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

scholarly journals The Picornavirus Avian Encephalomyelitis Virus Possesses a Hepatitis C Virus-Like Internal Ribosome Entry Site Element

2007 ◽  
Vol 82 (4) ◽  
pp. 1993-2003 ◽  
Author(s):  
Mehran Bakhshesh ◽  
Elisabetta Groppelli ◽  
Margaret M. Willcocks ◽  
Elizabeth Royall ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Secondary Structure ◽  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Hepatitis A Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Encephalomyelitis Virus ◽  
Ires Element

ABSTRACT Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotide-long 5′ untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3′ end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.

scholarly journals A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 42-50 ◽  
Author(s):  
RA Sokolic ◽  
S Sekhsaria ◽  
Y Sugimoto ◽  
N Whiting-Theobald ◽  
GF Linton ◽  
...  
Keyword(s):  
Gene Therapy ◽  
B Cells ◽  
Resistance Gene ◽  
Internal Ribosome Entry Site ◽  
Therapeutic Gene ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element ◽  
O2 Production

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

scholarly journals Unique Characteristics of a Picornavirus Internal Ribosome Entry Site from the Porcine Teschovirus-1 Talfan

2002 ◽  
Vol 76 (22) ◽  
pp. 11721-11728 ◽  
Author(s):  
Yoshihiro Kaku ◽  
Louisa S. Chard ◽  
Toru Inoue ◽  
Graham J. Belsham
Keyword(s):  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
New Class ◽  
Porcine Teschovirus ◽  
Ires Element

ABSTRACT 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.

scholarly journals 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

1998 ◽  
Vol 72 (7) ◽  
pp. 5638-5647 ◽  
Author(s):  
Saumitra Das ◽  
Michael Ott ◽  
Akemi Yamane ◽  
Weimin Tsai ◽  
Matthias Gromeier ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Internal Ribosome Entry Site ◽  
Hcv Infection ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element ◽  
Hcv Ires

ABSTRACT 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.

scholarly journals Functional Characterization of the X-Linked Inhibitor of Apoptosis (XIAP) Internal Ribosome Entry Site Element: Role of La Autoantigen in XIAP Translation

2000 ◽  
Vol 20 (13) ◽  
pp. 4648-4657 ◽  
Author(s):  
Martin Holcik ◽  
Robert G. Korneluk
Keyword(s):  
Internal Ribosome Entry Site ◽  
Physiological Stress ◽  
Present Report ◽  
Functional Characterization ◽  
Inhibitor Of Apoptosis ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
La Protein ◽  
Apoptosis Protein ◽  
Ires Element

ABSTRACT X-linked inhibitor of apoptosis protein (XIAP) is a key regulator of programmed cell death triggered by various apoptotic triggers. Translation of XIAP is controlled by a 162-nucleotide (nt) internal ribosome entry site (IRES) element located in the 5′ untranslated region of XIAP mRNA. XIAP IRES mediates efficient translation of XIAP under physiological stress and enhances cell protection against serum deprivation and radiation-induced apoptosis. In the present report we describe the assembly of a sequence-specific RNA-protein complex consisting of at least four cytosolic proteins on the XIAP IRES element. We determine that the core binding sequence is approximately 28 nt long and is located 34 nt upstream of the initiation site. Moreover, we identify the La autoantigen as a protein that specifically bindsXIAP IRES in vivo and in vitro. The biological relevance of this interaction is further demonstrated by the inhibition ofXIAP IRES-mediated translation in the absence of functional La protein. The results suggest an important role for the La protein in the regulation of XIAP expression, possibly by facilitating ribosome recruitment to the XIAP IRES.

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

2007 ◽  
Vol 88 (11) ◽  
pp. 3053-3062 ◽  
Author(s):  
Olga Fernández-Miragall ◽  
Encarnación Martínez-Salas
Keyword(s):  
Internal Ribosome Entry Site ◽  
Living Cells ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Essential Information ◽  
Mouth Disease Virus ◽  
Ires Element

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.

scholarly journals The 5′ Untranslated Region of Rhopalosiphum padi Virus Contains an Internal Ribosome Entry Site Which Functions Efficiently in Mammalian, Plant, and Insect Translation Systems

2001 ◽  
Vol 75 (21) ◽  
pp. 10244-10249 ◽  
Author(s):  
Kathryn E. Woolaway ◽  
Konstantinos Lazaridis ◽  
Graham J. Belsham ◽  
Michael J. Carter ◽  
Lisa O. Roberts
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rhopalosiphum Padi ◽  
Encephalomyocarditis Virus ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element ◽  
Translation Systems

ABSTRACT Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5′ UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5′ UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, andDrosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5′ IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.

Sign in / Sign up

Export Citation Format

Share Document