Cellular Proteins Act as Bridge Between 5′ and 3′ Ends of the Coxsackievirus B3 Mediating Genome Circularization During RNA Translation
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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.
KeywordsInternal Ribosome Entry Site Polypyrimidine Tract Binding Internal Ribosome Entry Site Element Internal Ribosome Entry Site Activity Viral Translation
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Conflict of interest
The authors declare that they have no conflict of interest.
- 5.Bhattacharyya S, Das S (2005) Mapping of secondary structure of the spacer region within the 5′-untranslated region of the Coxsackievirus B3 RNA: possible role of an apical GAGA loop in binding La protein and influencing internal initiation of translation. Virus Res 108:89–100PubMedCrossRefGoogle Scholar
- 12.Hinnebusch AG, Dever TE, Asano K (2007) Mechanism of translation initiation in the yeast Saccharomyces cerevisiae. In: Mathews MB, Sonenberg N, Hershey JWB (eds) Translational control in biology and medicine. Cold Spring Harbor Laboratory Press, New York, pp 225–268Google Scholar
- 39.Yang D, Wilson JE, Anderson DR, Bohunek L, Cordeiro C, Kandolf R, McManus BM (1997) In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5′ untranslated region of Coxsackievirus B3: potential targets for antiviral action of antisense oligomers. Virology 228:63–73PubMedCrossRefGoogle Scholar