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Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin.

ABSTRACT: Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.

SUBMITTER: Kazadi K

PROVIDER: S-EPMC2588522 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin.

Kazadi Kayole K Loeuillet Corinne C Deutsch Samuel S Ciuffi Angela A Muñoz Miguel M Beckmann Jacques S JS Moradpour Darius D Antonarakis Stylianos E SE Telenti Amalio A

Nucleic acids research 20081031 21

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and ...[more]

PMID: 18978018

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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.

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Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin.

Publications

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin.

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