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Souii A.,Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives LR99 ES27 | Ben M'hadheb-Gharbi M.,Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives LR99 ES27 | Ben M'hadheb-Gharbi M.,University of Monastir | Sargueil B.,University of Paris Descartes | And 5 more authors.
International Journal of Molecular Sciences | Year: 2013

Coxsackievirus B3 (CVB3) is an enterovirus of the family of Picornaviridae. The Group B coxsackieviruses include six serotypes (B1 to B6) that cause a variety of human diseases, including myocarditis, meningitis, and diabetes. Among the group B, the B3 strain is mostly studied for its cardiovirulence and its ability to cause acute and persistent infections. Translation initiation of CVB3 RNA has been shown to be mediated by a highly ordered structure of the 5′-untranslated region (5′UTR), which harbors an internal ribosome entry site (IRES). Translation initiation is a complex process in which initiator tRNA, 40S and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of the mRNA. We have previously addressed the question of whether the attenuating mutations of domain V of the poliovirus IRES were specific for a given genomic context or whether they could be transposed and extrapolated to a genomic related virus, i.e., CVB3 wild-type strain. In this context, we have described that Sabin3-like mutation (U473→C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In this study, we analyzed the efficiency of formation of ribosomal initiation complexes 48S and 80S through 10%-30% and 10%-50% sucrose gradients using rabbit reticulocyte lysates (RRLs) and stage-specific translation inhibitors: 5′-Guanylyl-imidodiphosphate (GMP-PNP) and Cycloheximide (CHX), respectively. We demonstrated that the interaction of 48S and 80S ribosomal complexes within the mutant CVB3 RNA was abolished compared with the wild-type RNA by ribosome assembly analysis. Taken together, it is possible that the mutant RNA was unable to interact with some trans-acting factors critical for enhanced IRES function. © 2013 by the authors; licensee MDPI, Basel, Switzerland. Source


Souii A.,Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives LR99 ES27 | Gharbi J.,Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives LR99 ES27 | Gharbi J.,University of Monastir | Ben M'hadheb-Gharbi M.,Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives LR99 ES27 | Ben M'hadheb-Gharbi M.,University of Monastir
International Journal of Molecular Sciences | Year: 2013

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, meningitis and pancreatitis. CVB3 overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap independent manner at the viral internal ribosomal entry site. The 5′ untranslated region (5′UTR) of CVB3 genomic RNA is highly structured. It is the site of multiple RNA-protein and RNA-RNA interactions and it plays a critical role during translation initiation. Similar to the 5′UTR, CVB3 3′ untranslated region (3′UTR) also contains secondary structural elements consisting of three stem-loops followed by a poly (A) tail sequence. Long-range RNA-RNA interactions between 5′ and 3′ ends of some viral genomes have been observed. Because of their dual role in translation and replication, the 5′ and 3′UTRs represent promising candidates for the study of CVB3 cardiovirulence. Taking into account that efficient initiation of mRNA translation depends on a temporally and spatially orchestrated sequence of protein-protein, protein-RNA and RNA-RNA interactions, and that, at present, little is known about RNA-RNA interactions between CVB3 5′ and 3′UTRs, we aimed in the present study, to assess a possible RNA-RNA interaction between 5′ and 3′UTRs during the initiation of translation of a wild-type and a previously characterized mutant (Sabin3-like) CVB3 strains and to investigate the effect of the Sabin3-like mutation on these potential interactions. For this purpose, "Electrophoretic Mobility Shift" assays were carried out. Data obtained did not show any RNA-RNA direct interactions between the 5′- and 3′- ends. Therefore, we can suggest that the possible mechanism by which 3′UTR enhances CVB3 IRES activity may be by bridging the 5′ to the 3′ end through RNA-protein interaction and not through RNA-RNA direct contact. However, these findings need to be confirmed by carrying out further experiments. © 2013 by the authors; licensee MDPI, Basel, Switzerland. Source

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