Cellular Proteins Act as Bridge Between 5′ and 3′ Ends of the Coxsackievirus B3 Mediating Genome Circularization During RNA Translation

Abstract

The positive single-stranded RNA genome of the Coxsackievirus B3 (CVB3) contains a 5′ untranslated region (UTR) which hosts the internal ribosome entry site (IRES) element that governs cap-independent translation initiation and a polyadenylated 3′ UTR which is required for stimulating the IRES activity. Viral RNA genomes could circularize to regulate initiation of translation and RNA synthesis at 5′ and 3′ ends. Interactions could either take place by direct RNA–RNA contacts, through cellular protein bridges mediating RNA circularization or both. Accordingly, we aimed to assess the nature of molecular interactions between these two regions and to evaluate cellular factors required for mRNA 3′ end-mediated stimulation of CVB3 IRES-driven translation. By gel shift assays, we have showed that combining, in vitro, 5′ and 3′ UTR fragments had no discernible effect on the structures of RNAs, arguing against the presence of specific canonical RNA–RNA cyclization sequences between these two regions. Competitive UV crosslinking assays using BHK-21 cell extract showed common cellular proteins eIF3b, PTB, and La binding to both 5′- and 3′ end RNAs. PCBP 1–2 and PABP were shown to bind, respectively, to 5′ and 3′ UTR probes. Taking together, these data suggest that CVB3 5′–3′ end bridging occurs through 5′ UTR–protein–protein–3′ UTR interactions and not through RNA–RNA direct contact. The dual involvement of the 3′ and 5′ UTRs in controlling viral translation and RNA synthesis highlights the relevance of these regions in the infectious virus life cycle, making them suitable candidates for targeted CVB3 antiviral therapy.