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 (U473 → 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.

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Figure 1

Electrophoretic profile, observed on 2% agarose gel, of CVB3 wild-type and Sabin3-like domain V DNAs. Lane MW: molecular weight DNA marker; lane (−): a negative control for the reaction; lane DV_(Wt): domain V wild-type DNA; lane DV_(S3): domain V Sabin3-like DNA. Experimental conditions were carried out as described in the “Materials and Methods” section.

Figure 2

Autoradiogram of an UV-crosslink assay performed with IRES domain V labeled probes and an extract prepared from HeLa cells. Proteins were resolved in a 10% SDS-PAGE. Domain V of FMDV IRES was used as positive control (lane DV FMDV). Lanes DV CVB3 _(Wt) and DV CVB3 _(S3) demonstrate the RNA–protein interaction pattern of CVB3 wild-type and Sabin3-like IRES domains V. Equal amounts of total proteins (10 μg) were loaded per well. The molecular weight (MW) of each complex was determined by comparing to a concurrently loaded MW marker: Prestained SDS-page standards broad range (Biorad).

Figure 3

UV-cross-linking to determine the molecular weight of RNA binding proteins to domain V of the CVB3 IRES. (α³²P)-CTP-labeled probes were added to BHK-21 cell total protein extracts and cross-linked by UV-light exposure, followed by RNase A treatment. Proteins that cross-linked to radioactive RNAs were detected by 10% SDS-PAGE and subsequent autoradiography. Lanes DV CVB3 _(Wt) and DV CVB3 _(S3) demonstrate protein-probe interactions within CVB3 wild-type and Sabin3-like domain V RNAs, respectively. Lane DV FMDV indicates RNA-protein complexes formed within the FMDV domain V. Equal amounts of total proteins (10 μg) were loaded per well. The molecular weight of each complex was determined by comparing to a concurrently loaded molecular weight (MW) marker: Prestained SDS-page standards broad range (Biorad).

Figure 4

Filter-binding assays of some initiation factors binding to CVB3 wild-type (Wt) and Sabin3-like (S3) Domains V (DV) RNAs. Purified initiation factors eIF4G (a), eIF4B (b), and eIF3 (c) directly bind to the wild-type and the mutant Domain V RNAs.