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首页> 外文期刊>Journal of Molecular Biology >An RNA domain within the 5 ' untranslated region of the tomato bushy stuntvirus genome modulates viral RNA replication
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An RNA domain within the 5 ' untranslated region of the tomato bushy stuntvirus genome modulates viral RNA replication

机译：番茄丛状特技病毒基因组5'非翻译区内的RNA结构域调节病毒RNA复制

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摘要

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted Sis, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (-)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5' terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 sub-element, in tombusvirus RNA replication.
机译：分析了番茄浓密特技病毒的（+）链RNA基因组中5'非翻译区（UTR）的末端一半在病毒RNA复制中的可能作用。计算机辅助二级结构的热力学分析，碱基对协变的系统发育比较以及化学和酶溶液结构探测被用于分析78个核苷酸长的5'-末端序列。结果表明该序列采用包含三螺旋结核的分支二级结构。由该末端序列形成的T形结构域（TSD）由一个突出的十个碱基对长螺旋（称为茎1（S1））封闭。在模型亚病毒RNA复制子中删除形成S1的5'或3'区段（分别为坐标1-10或69-78），即原型缺陷干扰（DI）RNA，可降低该分子在体内的积累水平20倍。在此复制子内对S1进行的补偿型突变分析表明，预测的S1结构的形成与有效的DI RNA积累之间存在很强的相关性。体内RNA衰变研究并未发现包含被破坏的Sis的DI RNA的物理稳定性有任何显着变化，因此暗示RNA复制是受影响的过程。进一步的研究表明，（+）链中S1的不稳定比（-）链不稳定对DI RNA积累的危害更大，因此S1介导的活性可能主要通过（+）链发挥作用。 S1在DI RNA积累中的重要作用促使我们研究了先前鉴定的突变DI RNA RNA B的5'-近端二级结构，该结构缺少5'UTR但仍具有低水平的复制能力。突变的5'末端TSD中存在的预测S1样元件的突变分析证实了前者在RNA B积累中的重要性。总体而言，这些数据支持TSD，尤其是其S1子元素在鼓膜病毒RNA复制中的基本作用。

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