PDF(Pubmed)
Abstract：
RNA structure in the influenza A virus (IAV) has been the focus of several studies that have shown connections between conserved secondary structure motifs and their biological function in the virus replication cycle. Questions have arisen on how to best recognize and understand the pandemic properties of IAV strains from an RNA perspective, but determination of the RNA secondary structure has been challenging. Herein, we used chemical mapping to determine the secondary structure of segment 8 viral RNA (vRNA) of the pandemic A/California/04/2009 (H1N1) strain of IAV. Additionally, this long, naturally occurring RNA served as a model to evaluate RNA mapping with 4-thiouridine (4sU) crosslinking. We explored 4-thiouridine as a probe of nucleotides in close proximity, through its incorporation into newly transcribed RNA and subsequent photoactivation. RNA secondary structural features both universal to type A strains and unique to the A/California/04/2009 (H1N1) strain were recognized. 4sU mapping confirmed and facilitated RNA structure prediction, according to several rules: 4sU photocross-linking forms efficiently in the double-stranded region of RNA with some flexibility, in the ends of helices, and across bulges and loops when their structural mobility is permitted. This method highlighted three-dimensional properties of segment 8 vRNA secondary structure motifs and allowed to propose several long-range three-dimensional interactions. 4sU mapping combined with chemical mapping and bioinformatic analysis could be used to enhance the RNA structure determination as well as recognition of target regions for antisense strategies or viral RNA detection.
摘要：
甲型流感病毒（IAV）中的RNA结构已成为几项研究的重点，这些研究表明保守的二级结构基序与其在病毒复制周期中的生物学功能之间存在联系。关于如何从RNA的角度最好地识别和理解IAV毒株的大流行特性，但RNA二级结构的确定一直具有挑战性。在这里，我们使用化学作图来确定IAV大流行A/California/04/2009(H1N1)株的第8段病毒RNA(vRNA)的二级结构.此外,这么长,天然存在的RNA用作评估4-硫尿苷（4sU）交联的RNA作图的模型。我们探索了4-硫尿苷作为紧密接近的核苷酸探针，通过掺入新转录的RNA和随后的光活化。RNA二级结构特征对于A型菌株是通用的并且对于A/California/04/2009(H1N1)菌株是独特的。4sU作图证实并促进了RNA结构预测，根据几个规则：4sU光交联在RNA的双链区域有效地形成，具有一定的灵活性，在螺旋的末端，并在允许其结构移动性的情况下穿过凸起和环路。该方法突出了片段8vRNA二级结构基序的三维特性，并允许提出几种长程三维相互作用。4sU作图结合化学作图和生物信息学分析可用于增强RNA结构确定以及对反义策略或病毒RNA检测的靶区域的识别。
