Dataset Information

Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors.

ABSTRACT:

Background

Polycistronic retroviral vectors that contain several therapeutic genes linked via internal ribosome entry sites (IRES), provide new and effective tools for the co-expression of exogenous cDNAs in clinical gene therapy protocols. For example, tricistronic retroviral vectors could be used to genetically modify antigen presenting cells, enabling them to express different co-stimulatory molecules known to enhance tumor cell immunogenicity.

Results

We have constructed and compared different retroviral vectors containing two co-stimulatory molecules (CD70, CD80) and selectable marker genes linked to different IRES sequences (IRES from EMCV, c-myc, FGF-2 and HTLV-1). The tricistronic recombinant amphotropic viruses containing the IRES from EMCV, FGF-2 or HTLV-1 were equally efficient in inducing the expression of an exogenous gene in the transduced murine or human cells, without displaying any cell type specificity. The simultaneous presence of several IRESes on the same mRNA, however, can induce the differential expression of the various cistrons. Here we show that the IRESes of HTLV-1 and EMCV interfere with the translation induced by other IRESes in mouse melanoma cells. The IRES from FGF-2 did however induce the expression of exogenous cDNA in human melanoma cells without any positive or negative regulation from the other IRESs present within the vectors. Tumor cells that were genetically modified with the tricistronic retroviral vectors, were able to induce an in vivo anti-tumor immune response in murine models.

Conclusion

Translation of the exogenous gene is directed by the IRES and its high level of expression not only depends on the type of cell that is transduced but also on the presence of other genetic elements within the vector.

SUBMITTER: Douin V

PROVIDER: S-EPMC514710 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Publications

Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors.

Douin Victorine V Bornes Stephanie S Creancier Laurent L Rochaix Philippe P Favre Gilles G Prats Anne-Catherine AC Couderc Bettina B

BMC biotechnology 20040727

<h4>Background</h4>Polycistronic retroviral vectors that contain several therapeutic genes linked via internal ribosome entry sites (IRES), provide new and effective tools for the co-expression of exogenous cDNAs in clinical gene therapy protocols. For example, tricistronic retroviral vectors could be used to genetically modify antigen presenting cells, enabling them to express different co-stimulatory molecules known to enhance tumor cell immunogenicity.<h4>Results</h4>We have constructed and c ...[more]

PMID: 15279677

Similar Datasets

Project description:In contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system in Cryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimized Renilla and firefly luciferase (ORluc and OFluc, respectively) gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5' untranslated regions (UTRs) of the genomes of diverse positive-sense single-stranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5'-proximal codons of CHV1 as well as the 5' UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi.IMPORTANCE Cap-independent, internal ribosomal entry site (IRES)-mediated translational initiation is often used by virus mRNAs and infrequently by cellular mRNAs in animals and plants. However, no IRESs have been established in fungal virus RNAs or cellular RNAs in filamentous fungi. Here, we report the development of a dual-luciferase assay system and measurement of the IRES activities of fungal RNA viruses in a model filamentous fungal host, Cryphonectria parasitica Viruses identified as IRES positive include hypoviruses (positive-sense RNA viruses, members of the expanded Picornavirus supergroup), totiviruses (nonsegmented dsRNA viruses), and chrysoviruses (tetrasegmented dsRNA viruses). No IRES activities were observed in the 5' untranslated regions of mycoreoviruses (11-segmented dsRNA viruses), quadriviruses (tetrasegmented dsRNA viruses), or partitiviruses (bisegmented dsRNA viruses). This study provides the first evidence for IRES activities in diverse RNA viruses in filamentous fungi and is a first step toward identifying trans-acting host factors and cis-regulatory viral RNA elements.

Dataset Information

Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors.

Background

Results

Conclusion

Publications

Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors.

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