Research Unit UR17ES30 "Virology & Antiviral Strategies", Institute of Biotechnology, University of Monastir, Tahar HADDED Street, 5000, Monastir, Tunisia.
Department of Biological Sciences, College of Science, King Faisal University, P.O. Box 380, Al-Ahsa, 31982, Saudi Arabia.
Curr Microbiol. 2022 Aug 16;79(10):288. doi: 10.1007/s00284-022-02986-3.
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.
柯萨奇病毒 B3（CVB3）基因组 RNA 的 5'UTR 异常长且富含高度结构化的二级结构，包含一个保守的顺式作用 RNA 元件，名为隐匿 AUG（cAUG），细胞 48S 复合物在此形成。在本研究中，我们研究了该 cAUG 在 CVB3 翻译、复制和毒力中的作用。通过定点突变构建了突变型病毒亚基因组复制子 RNA。我们比较了 cAUG 突变体与野生型菌株在体外翻译和复制效率以及体内毒力方面的特征。分别使用 UV 交联测定和实时 PCR 来检测与宿主蛋白的结合和病毒产生的定量。研究并比较了包含 cAUG 位点的结构域的二级结构。结果表明，CVB3 5'UTR 中的引入突变影响了体外和体内病毒翻译，无法通过补偿突变来挽救。揭示了 La 和 PCBP2 翻译起始因子与突变体 cAUG 残基的相互作用减少。还证明了病毒突变体 RNA 产生减少。此外，二级结构预测揭示了突变体 sense 链 RNA 的 cAUG 部分的核糖体结合位点发生变化，而野生型的结构没有改变，这表明 cAUG 突变特异性影响了 sense RNA 链的二级结构。总之，AUG 的完整性通过 IRES 元件影响核糖体募集的效率和复制的能力。
