Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Nucleic Acids Res. 2022 Sep 23;50(17):10078-10092. doi: 10.1093/nar/gkac738.
Due to genome segmentation, rotaviruses must co-package eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus NSP2 protein. While the activities of distinct RNA chaperones are well studied on smaller RNAs, little is known about their global effect on the entire viral transcriptome. Here, we used Selective 2'-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to examine the secondary structure of the rotavirus transcriptome in the presence of increasing amounts of NSP2. SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate rotavirus transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of mutation rates measured by mutational profiling reveals a global 5-fold rate increase in the presence of NSP2. We demonstrate that the normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone. Analysis of the mutation rates reveals a larger effect on stems rather than loops. Together, these data provide the first experimentally derived secondary structure model of the rotavirus transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner.
由于基因组的分段，轮状病毒必须共同包装十一个不同的基因组 RNA。这种包装是由病毒编码的 RNA 伴侣介导的，例如轮状病毒 NSP2 蛋白。虽然不同 RNA 伴侣的活性在较小的 RNA 上得到了很好的研究，但它们对整个病毒转录组的全局影响知之甚少。在这里，我们使用选择性 2'-羟基乙酰化分析引物延伸和突变分析 (SHAPE-MaP) 来研究在增加 NSP2 量的情况下轮状病毒转录本的二级结构。SHAPE-MaP 数据表明，尽管 NSP2 在体外具有很好的解链活性，但它与同源轮状病毒转录本孵育不会导致 SHAPE 反应性发生显著变化。然而，通过突变分析测量的突变率的定量分析揭示了在存在 NSP2 的情况下，整体速率增加了 5 倍。我们证明，从突变率推导出 SHAPE 反应性的归一化程序可能掩盖了 RNA 伴侣的重要全局效应。突变率的分析显示，对茎而不是环的影响更大。这些数据共同提供了轮状病毒转录组的第一个实验衍生的二级结构模型，并揭示了 NSP2 通过以浓度依赖的方式全局增加 RNA 骨架的灵活性来发挥作用。
