Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA.
Department of Biomedical Engineering and Genome Center, University of California Davis, Davis, CA, USA.
Nucleic Acids Res. 2018 Mar 16;46(5):2573-2584. doi: 10.1093/nar/gkx1273.
In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3' untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites.
在单链 (+) 义 RNA 病毒中，RNA 结构元件 (SEs) 在从复制到包装的感染过程中发挥着重要作用。我们使用选择性 2'-羟乙酰化分析通过引物延伸测序 (SHAPE-Seq) 和变异性分析，探索黄瓜花叶病毒 (CMV) 第三个基因组片段 RNA3（2216 个核苷酸）的结构特征，无论是在体外还是在植物细胞裂解物中。比较 SHAPE-Seq 和变异性分析结果显示，衣壳蛋白开放阅读框和 3'非翻译区存在多个 SEs。其中四个 SEs 发生突变，并在烟草植物中连续传代，以鉴定对原始突变序列的生物选择变化。传代后，环突变体显示出部分回复到其野生型序列，并且通过植物体内同义突变恢复到野生型样结构的突变破坏的 SEs。这些结果支持病毒开放阅读框 SEs 在宿主生物体内的存在和选择，并为进一步研究 RNA 结构在病毒感染中的作用提供了框架。此外，这项工作证明了高通量化学探测在植物细胞裂解物中的适用性，并提出了一种从重叠逆转录酶启动子位点计算 SHAPE 反应性的新方法。
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