In Vitro Molecular Characterization of RNA–Proteins Interactions During Initiation of Translation of a Wild-Type and a Mutant Coxsackievirus B3 RNAs

Abstract

Translation initiation of Coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. Host cell factors involved in this process include some canonical translation factors and additional RNA-binding proteins. We have, previously, described that the Sabin3-like mutation (U475 → C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. With the aim to identify proteins interacting with CVB3 wild-type and Sabin3-like IRESes and to study interactions between HeLa cell or BHK-21 protein extracts and CVB3 RNAs, UV-cross-linking assays were performed. We have observed a number of proteins that specifically interact with both RNAs. In particular, molecular weights of five of these proteins resemble to those of the eukaryotic translation initiation factors 4G, 3b, 4B, and PTB. According to cross-linking patterns obtained, we have demonstrated a better affinity of CVB3 RNA binding to BHK-21 proteins and a reduced interaction of the mutant RNA with almost cellular polypeptides compared to the wild-type IRES. On the basis of phylogeny of some initiation factors and on the knowledge of the initiation of translation process, we focused on the interaction of both IRESes with eIF3, p100 (eIF4G), and 40S ribosomal subunit by filter-binding assays. We have demonstrated a better affinity of binding to the wild-type CVB3 IRES. Thus, the reduction efficiency of the mutant RNA to bind to cellular proteins involved in the translation initiation could be the reason behind inefficient 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

“…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]. …”

“…The identity of these proteins was then confirmed by immunoprecipitation. A third band migrating at an apparent molecular mass of about 220 kDa, as expected for eIF4G [22,43] was also shown to bind, specifically, with CVB3 transcripts. Additionally, several other bands were labeled indicating the presence of other proteins binding to both viral IRES domains V. The smear-like patterns suggest the presence of multiple RNA–protein complexes over a range of molecular weights.…”

“…In order to analyze the cloned sequences, randomly chosen clones were tested by PCR-colony as previously described [43]. Positive clones were, then, sequenced using an ABI Prism BigDye Terminators Sequencing Kit (Applied Biosystems).…”

“…The poor translation efficiency of the Sabin3-like IRES was then explicated by its inability to correctly bind some essential non-canonical translation factors during the initiation of translation. 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.…”

“…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.…”