Impaired binding of standard initiation factors eIF3b, eIF4G and eIF4B to domain V of the live-attenuated coxsackievirus B3 Sabin3-like IRES - alternatives for 5′UTR-related cardiovirulence mechanisms

ABSTRACT: Internal ribosome entry site (IRES) elements fold into highly organized conserved secondary and probably tertiary structures that guide the ribosome to an internal site of the RNA at the IRES 3′end. The composition of the cellular proteome is under the control of multiple processes, one of the most important being translation initiation. In each poliovirus Sabin vaccine strain, a single point mutation in the IRES secondary-structure domain V is a major determinant of neurovirulence and translation attenuation. Here we are extrapolating poliovirus findings to a genomic related virus named coxsackievirus B3 CVB3); a causative agent of viral myocarditis. We have previously reported that Sabin3-like mutation (U(473) → C) introduced in the domain V sequence of the CVB3 IRES led to a defective mutant with a serious reduction in translation efficiency and ribosomal initiation complex assembly, besides an impaired RNA-protein binding pattern. With the aim to identify proteins interacting with both CVB3 wild-type and Sabin3-like domain V RNAs and to assess the effect of the Sabin3-like mutation on these potential interactions, we have used a proteomic approach. This procedure allowed the identification of three RNA-binding proteins interacting with the domain V: eIF4G (p220), eIF3b (p116) and eIF4B (p80). Moreover, we report that this single-nucleotide exchange impairs the interaction pattern and the binding affinity of these standard translation initiation factors within the IRES domain V of the mutant strain. Taken together, these data indicate how this decisive Sabin3-like mutation mediates viral translation attenuation; playing a key role in the understanding of the cardiovirulence attenuation within this construct. Hence, these data provide further evidence for the crucial role of RNA structure for the IRES activity, and reinforce the idea of a distribution of function between the different IRES structural domains. VIRTUAL SLIDE: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6160165131045880

Molecular Analysis of RNA-RNA Interactions between 5’ and 3’ Untranslated Regions during the Initiation of Translation of a Cardiovirulent and a Live-Attenuated Coxsackievirus B3 Strains

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

Ribosomal Initiation Complex Assembly within the Wild-Strain of Coxsackievirus B3 and Live-Attenuated Sabin3-like IRESes during the Initiation of Translation

Abstract: 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 thisInt. J. Mol. Sci. 2013, 14 440