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

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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.

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Acknowledgements

The Authors acknowledge the Tunisian Ministry of Higher Education and Scientific Research MESRS (Research Unit UR17ES30) and the Indian Department of Science and Technology DST (Indian Institute of Science).

Funding

This work was funded by the Indian international collaboration research project [Grant Nb. INT/P-09/2013]. This work was also supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant Nb. 280].

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Authors and Affiliations

  1. Research Unit UR17ES30 “Virology & Antiviral Strategies”, Institute of Biotechnology, University of Monastir, Tahar HADDED Street, 5000, Monastir, Tunisia

    Ahlem Ben Youssef & Manel Ben M’hadheb

  2. Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa, 31982, Saudi Arabia

    Jawhar Gharbi

  3. Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India

    Biju George & Saumitra Das

Authors
  1. Ahlem Ben Youssef
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  2. Jawhar Gharbi
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  3. Biju George
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  4. Saumitra Das
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  5. Manel Ben M’hadheb
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Contributions

ABY and BG realized all experiments and write article. JG, SD and MBM designed the work and review submission.

Corresponding author

Correspondence to Manel Ben M’hadheb.

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All authors declared that they have no conflict of interest.

Ethical Approval

The animal model study in this work was carried out in strict accordance with the recommendations of the Committee of Ethics of the Institute of Biotechnology of Monastir described in their guide for the use of experimentations with animal models. The protocol was controlled and supervised by of a member of the committee. Animal experiences were conducted with making all efforts to minimize suffering animals during anesthesia. Mice controls were bred in the Biotechnology Institute of Monastir animal facility. All animal experiments were in compliance with the recommendations of the Committee of Ethics of the institution in a protocol approved and supervised by the committee (ISBM 2021/0015).

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Ben Youssef, A., Gharbi, J., George, B. et al. A Single Mutation in the Cryptic AUG (cAUG) Affects In Vitro Translation and Replication Efficiencies and In Vivo Virulence of Coxsackievirus B3 (CVB3). Curr Microbiol 79, 288 (2022). https://doi.org/10.1007/s00284-022-02986-3

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  • DOI: https://doi.org/10.1007/s00284-022-02986-3

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