IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector.
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The internal ribosome entry site (IRES) has been widely used to coexpress heterologous gene products by a message from a single promoter. However, little is known about the efficiency of IRES-dependent second gene expression in comparison with that of first gene expression. This study was undertaken to characterize the relative expression of IRES-dependent second gene in a bicistronic vector, which was derived from the 5' untranslated regions of the encephalomyocarditis virus (EMCV). IRES-dependent second gene expression was compared with cap-dependent first gene expression in several cultured cell lines and in mouse liver in vivo. The expression of the IRES-dependent second gene ranged from 6 to 100% (in most cases between 20 and 50%) that of the first gene. Second gene expression in a plasmid without the IRES was 0.1-0.8% (with some exceptions) that of the first gene. These findings have important implications for the use of IRES, i.e., care should be taken regarding the decreased capacity of IRES-dependent downstream gene expression as well as in determining which gene should be positioned as the first or second gene in a bicistronic vector. (+info)
Inherent instability of poliovirus genomes containing two internal ribosome entry site (IRES) elements supports a role for the IRES in encapsidation.
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Previous studies have described poliovirus genomes in which the internal ribosome entry (IRES) for encephalomyocarditis virus (EMCV) is positioned between the P1 and P2-P3 open reading frames of the poliovirus genome. Although these dicistronic poliovirus genomes were replication competent, most exhibited evidence of genetic instability, and the EMCV IRES was deleted upon serial passage. One possible reason for instability of the genome is that the dicistronic genome was at least 108% larger than the wild-type poliovirus genome, which could reduce the efficiency of encapsidation. To address this possibility, we have constructed dicistronic poliovirus replicons by substituting the EMCV IRES and the gene encoding luciferase in place of the poliovirus P1 region; the resulting dicistronic replicons are smaller than the wild-type poliovirus genome. One dicistronic genome was constructed in which the poliovirus 5' nontranslated region was fused to the gene encoding luciferase, followed by the complete EMCV IRES fused to the P2-P3 region of the poliovirus genome (PV-Luc-EMCV). A second dicistronic genome, EMCV-Luc-PV, was constructed with the first 108 nucleotides of the poliovirus genome fused to the EMCV IRES, followed by the gene encoding luciferase and the poliovirus IRES fused to the remaining P2-P3 region of the poliovirus genome. Both dicistronic replicons expressed abundant luciferase following transfection of in vitro-transcribed RNA into HeLa cells at 30, 33, or 37 degrees C. The luciferase activity detected from PV-Luc-EMCV increased rapidly during the first 4 h following transfection and then plateaued, peaking after approximately 24 h. In contrast, the luciferase activity detected from EMCV-Luc-PV increased for approximately 12 h following transfection; by 24 h posttransfection, the overall levels of luciferase activity were similar to that of PV-Luc-EMCV. To analyze encapsidation of the dicistronic replicons, we used a system in which the capsid protein (P1) is provided in trans from a recombinant vaccinia virus (VV-P1). The PV-Luc-EMCV replicon was unstable upon serial passage in the presence of VV-P1, with deletions of the EMCV IRES region detected even during the initial transfection at 37 degrees C. Following serial passage in the presence of VV-P1 at 33 or 30 degrees C, we detected deleted genomes in which the luciferase gene was fused with the P2-P3 genes of the poliovirus genome so as to maintain the translational reading frame. In contrast, the EMCV-Luc-PV replicon was genetically stable during passage with VV-P1 at 33 or 30 degrees C. The encapsidation of EMCV-Luc-PV was compared to that of monocistronic replicons encoding luciferase with either a poliovirus or EMCV IRES. Analysis of the encapsidated replicons after four serial passages with VV-P1 revealed that the dicistronic replicon was encapsidated more efficiently than the monocistronic replicon with the EMCV IRES but less efficiently than the monicistronic replicon with the poliovirus IRES. The results of this study suggest a genetic predisposition for picornavirus genomes to contain a single IRES region and are discussed with respect to a role of the IRES in encapsidation. (+info)
Prevention of encephalomyocarditis virus-induced diabetes by live recombinant Mycobacterium bovis bacillus Calmette-Guerin in susceptible mice.
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The D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct cytolysis of pancreatic beta-cells. cDNA covering the major outer capsid protein (VP1) of the EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG). None of the SJL/J mice immunized with live recombinant BCG-VP1 (rBCG-VP1) became diabetic when challenged with the highly diabetogenic EMC-D virus, but the control mice inoculated with normal BCG developed diabetes during the same challenge. VP1-specific antibodies (including neutralizing antibodies) were markedly increased over time and reached the maximum titer at week 10 after a single immunization. The plateau of the titer lasted longer than 4 weeks. Mice and guinea pigs immunized with live rBCG-VP1 showed strong delayed-type hypersensitivity to the VP1 of the EMC-D virus. The preventive immunity still worked effectively 10 months after the primary immunization. At that time, the VP1-specific antibody was almost undetectable in the bloodstream, but a large number of VP1-specific lymphocytes was found in the spleen of the immunized mice. Our results show that live rBCG-VP1 elicits effective humoral and long-lasting cellular immune responses against EMC-D virus infection that results in the prevention of virus-induced diabetes in susceptible mice. (+info)
Retroviral vectors containing a variant dihydrofolate reductase gene for drug protection and in vivo selection of hematopoietic cells.
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Transfer of drug resistance genes to hematopoietic cells is being studied as a means to protect against the myelosuppression associated with cancer chemotherapy and as a strategy for the in vivo selection and amplification of genetically modified cells. The goal of this study was to test if retroviral-mediated gene transfer of a dihydrofolate reductase (DHFR) variant (L22Y) could be used for in vivo selection of transduced myeloid cells and to determine what proportion of transduced cells was required for protection from myelosuppression. Based on previous work suggesting that selection with antifolates may also require inhibition of nucleoside transport mechanisms, mice transplanted with DHFR-transduced bone marrow cells were treated with trimetrexate and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine riboside phosphate. In vivo selection of transduced myeloid progenitors was seen in the bone marrow and in circulating mature peripheral blood cells following drug treatment. These results show that the novel combination of the L22Y-DHFR cDNA, trimetrexate and nitrobenzylmercaptopurine riboside phosphate can be used to select for transduced myeloid cells, and that this approach warrants further study in large animal models. A bicistronic vector containing a human CD24 reporter gene was used to determine the number of modified cells needed for chemoprotection. Partial protection from neutropenia was seen when greater than 10% of myeloid cells expressed the vector, and high levels of protection were obtained when the proportion exceeded 30%. These results suggest that gene transfer may be useful for myeloprotection in certain pediatric cancers, but that more efficient gene transfer will be required to apply this approach to adult cancer patients. (+info)
Development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression.
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Rubella virus (RUB) is a small plus-strand RNA virus classified in the Rubivirus genus of the family Togaviridae. Live, attenuated RUB vaccines have been successfully used in vaccination programs for over 25 years, making RUB an attractive vaccine vector. In this study, such a vector was constructed using a recently developed RUB infectious cDNA clone (Robo). Using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicated to produce a recombinant construct (termed dsRobo) that expressed reporter genes such as chloramphenicol acetyltransferase and green fluorescent protein (GFP) under control of the second subgenomic promoter. However, expression of the reporter genes, as exemplified by GFP expression by dsRobo/GFP virus, was unstable during passaging, apparently due to homologous recombination between the subgenomic promoters leading to deletion of the GFP gene. To improve the stability of the vector, the internal ribosome entry site (IRES) of a picornavirus, encephalomyocarditis virus, was used instead of the second subgenomic promoter to eliminate homology. Construction was initiated by first replacing the subgenomic promoter in the parent Robo infectious clone with the IRES. Surprisingly, viable virus resulted; this virus did not synthesize a subgenomic RNA. The subgenomic promoter was then reintroduced in an orientation such that a single subgenomic RNA was produced, GFP was the initial gene on this RNA, while the RUB structural protein open reading frame was downstream and under control of the IRES element. GFP expression by this vector was significantly improved in comparison to dsRobo/GFP. This strategy should be applicable to increase the stability of other togavirus vectors. (+info)
Immunoglobulin treatment ameliorates murine myocarditis associated with reduction of neurohumoral activity and improvement of extracellular matrix change.
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OBJECTIVES: We examined effects of immunoglobulin on murine myocarditis induced by encephalomyocarditis virus, not pathogenic to humans, and analyzed the plasma cytokine and catecholamine levels and the changes of the extracellular matrix with or without the treatment. BACKGROUND: We have previously shown that immunoglobulin therapy suppressed murine coxsackievirus B3 myocarditis by an antiviral effect. However, it is not yet determined whether beneficial effects of immunoglobulin for myocarditis are due to antiviral effects or to other unknown effects. METHODS: Antiviral activity of human immunoglobulin (Polyglobin-N) against encephalomyocarditis virus was determined in vitro. Immunoglobulin (1 g/kg/day) was administered intraperitoneally to the virus-infected mice daily for two weeks, beginning simultaneously with virus inoculation in experiment I and on day 14 after virus inoculation in experiment II. RESULTS: Antiviral activity of immunoglobulin could not be detected in the assay of a plaque-reduction method in vitro. The in vivo study showed that immunoglobulin administration ameliorated both myocardial necrosis with interstitial fibrin deposition in experiment I and interstitial fibrosis with the improvement of ventricular remodeling in experiment II by the reduction of plasma catecholamines, interferon-alpha, and soluble intercellular adhesion molecule-1. CONCLUSIONS: Immunoglobulin therapy could suppress myocarditis associated with the improvement of extracellular matrix changes by the reduction of neurohumoral activity. (+info)
Activation of NF-kappaB by double-stranded RNA (dsRNA) in the absence of protein kinase R and RNase L demonstrates the existence of two separate dsRNA-triggered antiviral programs.
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Double-stranded RNA (dsRNA) of viral origin triggers two programs of the innate immunity in virus-infected cells. One is intended to decrease the rate of host cell protein synthesis and thus to prevent viral replication. This program is mediated by protein kinase R (PKR) and by RNase L and contributes, eventually, to the self-elimination of the infected cell via apoptosis. The second program is responsible for the production of antiviral (type I) interferons and other alarmone cytokines and serves the purpose of preparing naive cells for the viral invasion. This second program requires the survival of the infected cell and depends on the expression of antiapoptotic genes through the activation of the NF-kappaB transcription factor. The second program therefore relies on ongoing transcription and translation. It has been proposed that PKR plays an essential role in the activation of NF-kappaB by dsRNA. Here we present evidence that the dsRNA-induced NF-kappaB activity and the expression of beta interferon and inflammatory cytokines do not require either PKR or RNase L. Our results indicate, therefore, that the two dsRNA-activated programs are separate and can function independently of each other. (+info)
Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system.
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In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during initiation. We have developed and characterized a cell-free system from human HeLa cells that recapitulates this important feature, displaying more than one order of magnitude of translational synergism between the cap structure and the poly(A) tail. The stimulation of cap-dependent translation by the poly(A) tail is length-dependent, but not mediated by changes in mRNA stability. Using this system, we investigated the effect of the poly(A) tail on the translation of picornaviral RNAs, which are naturally polyadenylated but initiate translation via internal ribosome entry sites (IRESs). We show that translation driven by the IRESs of poliovirus (PV), encephalomyocarditis virus (EMCV), and hepatitis A virus is also significantly augmented by a poly(A) tail, ranging from an approximately 3-fold stimulation for the EMCV-IRES to a more than 10-fold effect for the PV IRES. These results raise interesting questions concerning the underlying molecular mechanism(s). The cell-free system described here should prove useful in studying these questions as well as providing a general biochemical tool to examine the translation initiation pathway in a more physiological setting. (+info)