The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing
The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing
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DOI：
10.1261/rna.89306
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发表时间：
2006-08-01
期刊：
影响因子：
4.5
通讯作者：
Casey, John L.
中科院分区：
文献类型：
--
作者：
Linnstaedt, Sarah D.;Kasprzak, Wojciech K.;Casey, John L.
RNA编辑在丁型肝炎病毒（HDV）的生命周期中起着关键作用。宿主编辑酶ADAR1识别HDV抗基因组中琥珀/W位点周围的特定RNA二级结构特征，并脱氨琥珀/W腺苷。先前的一份报告表明，分支二级结构是编辑HDV基因型III所必需的。这种分支结构不同于HDV复制所需的无分支棒状结构，目前只对其进行了部分表征，而且对其形成和稳定性的了解有限。在这里，我们研究了HDV基因型III RNA的二级结构、构象动力学和琥珀/W位点编辑，使用一种小型化的HDV基因型III RNA。使用mpgfold算法对该RNA进行计算分析表明，该RNA具有形成亚稳和稳定的非支化二级结构的倾向。此外，天然聚丙烯酰胺凝胶电泳表明，该RNA在体外转录时形成支链和非支链棒状结构。正如预测的那样，支链结构是一种亚稳结构，很容易转化为无支链的棒状结构。在体外，ADAR1只在琥珀/W位点编辑分支RNA。HDV基因型III RNA的结构异质性是显著的，因为不仅RNA的两种构象对病毒复制都具有重要的功能，而且两种形式的比例可以通过决定可用于修饰的底物RNA的数量来调节编辑。
RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification.