A Single Mutation in the Cryptic AUG (cAUG) Affects In Vitro Translation and Replication Efficiencies and In Vivo Virulence of Coxsackievirus B3 (CVB3)

Ben Youssef, A and Gharbi, J and George, B and Das, S and Ben M’hadheb, M (2022) A Single Mutation in the Cryptic AUG (cAUG) Affects In Vitro Translation and Replication Efficiencies and In Vivo Virulence of Coxsackievirus B3 (CVB3). In: Current Microbiology, 79 (10).

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Abstract

The 5′UTR of the genomic RNA of CVB3, unusually long and rich on highly structured secondary structure, contains a conserved cis acting RNA element named the cryptic AUG (cAUG), where the cellular 48S complex is formed. In this study, we investigate the role of this cAUG in CVB3 translation, replication, and virulence. Mutant viral sub-genomic replicon RNA was constructed by site-directed mutagenesis. We characterize in vitro translation and replication efficiencies and in vivo virulence of a cAUG mutant in comparison with wild-type strain. UV-cross-linking assay and Real-Time PCR were used, respectively, to detect binding host proteins and to quantify viral production. Secondary structures of domain containing the cAUG site were studied and compared. The results suggest that introduced mutation in the CVB3 5′UTR affects in vitro and ex vivo viral translation which cannot be rescued by compensatory mutations. A reduced interaction of the La and PCBP2 translation initiation factors with cAUG residue of mutant was revealed. Decreasing production of viral mutant RNA was also demonstrated. Furthermore, secondary structure prediction reveals changes in the ribosome binding sites of the cAUG moiety of mutant sense strand RNA and no alterations in the structure of wild type, suggesting that cAUG mutation specifically affects the secondary structure of the sense RNA strand. Taken together, AUG integrity influences the efficiency of ribosome recruitment through IRES element and the capacity of replication.

Item Type:	Journal Article
Publication:	Current Microbiology
Publisher:	Springer
Additional Information:	The copyright for this article belongs to the Springer.
Keywords:	PCBP2 protein, human; RNA binding protein; virus RNA, 5' untranslated region; conformation; Enterovirus B; genetics; HeLa cell line; human; metabolism; mutation; protein synthesis; virulence; virus replication, 5' Untranslated Regions; Enterovirus B, Human; HeLa Cells; Humans; Mutation; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Viral; RNA-Binding Proteins; Virulence; Virus Replication
Department/Centre:	Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited:	06 Oct 2022 10:41
Last Modified:	06 Oct 2022 10:41
URI:	https://eprints.iisc.ac.in/id/eprint/77179

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