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

Histology of hearts and pancreas of Swiss mice at day 10 after oral inoculation with CVB3 wild type and mutant viruses as described previously [26].

Shown are murine heart (a) and pancreas (b) tissues stained with hematoxylin and eosin from mice inoculated with CVB3 wild type, (c, d) heart and pancreas from a control uninfected mice and (e, f) heart and pancreas from mice inoculated with M3 virus (all 4 mutant viruses induced a similar lack of pathology in mice, S1 Fig). Lymphocyte infiltrations are shown in each organ tissues. Experiments were performed on three mice for each mutant and for each day post inoculation.</p

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

Characterization of Coxsackievirus B4 virus-like particles VLP produced by the recombinant baculovirus-insect cell system expressing the major capsid protein

Coxsackievirus B4 (CV-B4) is suspected to be an environmental factor that has the intrinsic capacity to damage the pancreatic beta cells and therefore causes insulitis and type 1 diabetes (T1D). Although vaccination against CV-B4 could reduce the incidence of this chronic auto-immune disease, there is currently no therapeutic reagent or vaccine in clinical use. By the employment of the Bac-to-Bac® vector system to express the major viral capsid protein, we contributed towards the development of a CV-B4 vaccine by producing CV-B4 virus-like particles (VLPs) from recombinant baculovirus in infected insect cells. In fact Western blot and Immunofluorescence analysis detected the viral protein 1 (VP1) in the cells resulting from the construction of a recombinant bacmid DNA carrying the key immunogenic protein then transfected in the insect cells. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained the recombinant protein and the electron microscopy demonstrated the presence of VLPs in these sucrose fractions. This study clearly shows for the first time the expression of CVB4 VP1 structure protein alone can form VLPs in the baculovirus-infected insect cell keeping conserved both characteristics and morphology.Authors acknowledge the Tunisian Ministry of Higher Education and Scientific Research (UR17ES30) and the Deanship of Scientific Research at King Faisal University for the financial support under Nasher Track (Grant No. 186390)