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 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.

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

“…Interestingly, the mutant RNA showed a reduced protein-binding profile compared to the wild-type domain V. A number of proteins binding to the domain V were identified: p220 (eIF4G), p116 (eIF3) and p80 (eIF4B). Filter-binding assays showed a better binding affinity of eIF3, eIF4G and eIF4B to the wild-type CVB3 domain V. These results perfectly correlate with the impaired protein-binding and the reduced translation efficiency previously reported with the Sabin3-like construct [ 41 - 44 ].…”

“…According to our previously published data [ 43 , 44 ], we suspected that the Sabin3-like mutation would impair the binding of cellular protein factors to the viral RNA that mediate the association of the ribosomal 40S subunit within the CVB3 IRES, and particularly, to the domain V. Four polypeptides of 220, 116, 80 and 57 kDa suspected as eIF4G, eIF3b, eIF4B and PTB, respectively bound to the CVB3 IRES RNA in the presence of BHK-21 cell extract and a reduction in the RNA-protein binding profile for the mutant RNA compared to the wild-type IRES were reported [ 43 ].…”

“…Our previous observations that the Sabin3-like point mutation in domain V of the CVB3 IRES seriously affected ribosome association [ 44 ] and protein-binding [ 43 ] within the full IRES sequence, coupled with the fact that domain V is the major determinant for binding of some initiation factors led us to the conclusion that this single nucleotide exchange may directly affect the binding of these translation initiation factors and hence impair the association of the ribosome with the viral RNA. In order to confirm this hypothesis, UV cross-link assays were performed.…”

“…To fill this gap, we have used a proteomic approach that has allowed the identification of some RNA-binding proteins interacting with both wild-type and mutant CVB3 IRESes [ 43 ]. Interestingly, the mutant RNA showed a reduced RNA-protein binding profile [ 43 ] and a reduced efficiency of ribosomal complex assembly [ 44 ] compared to the wild-type IRES. Taken together, these data were explicated by the inability of the mutant RNA to interact with some trans-acting factors critical for enhanced IRES function.…”

Inoculation of the attenuated Coxsackievirus B3 Sabin3-like strain induces a protection against virulent CVB3 Nancy and CVB4 E2 strains in Swiss mice by both oral and intraperitoneal routes

“…Accordingly, Ben M'hadheb- Gharbi et al ( , 2007 constructed equivalent attenuating mutations into the genomic context of CVB3 strains and reported that the Sabin3-like mutation (U 473 →C) led to a serious impairment of the translation efficiency (Souii et al 2013), a reduction of the virus titer and a destabilization of the secondary structure of the IRES. They also demonstrated that the inoculation, by oral route, of Swiss mice within the Sabin3-like strain did not show any histological alterations in heart tissue when compared to hearts of mice infected with the wild-CVB3 strain (Souii et al 2013).…”