Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements.
Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements.
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DOI：
10.1093/nar/gkx1273
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发表时间：
2018-03-16
影响因子：
14.9
通讯作者：
Thompson JR
中科院分区：
文献类型：
--
作者：
Watters KE;Choudhary K;Aviran S;Lucks JB;Perry KL;Thompson JR
在单链（+）-正义RNA病毒中，RNA结构元件（SE）在从复制到折叠的感染过程中起重要作用。利用引物延伸测序（SHAPE-Seq）和共变异分析技术，对黄瓜花叶病毒（CMV）基因组第3个片段RNA 3（2216 nt）的2′-羟基酰化进行了研究。SHAPE-Seq分析结果与协变分析结果比较，在外壳蛋白开放阅读框和3′端非翻译区发现多个SE。将这些SE中的四个突变并在烟草植物中连续传代，以鉴定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，并且在结构上被突变破坏的SE通过植物中的同义突变恢复到野生型样结构。这些结果支持了病毒开放阅读框SE在宿主生物中的存在和选择，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，并提出了一种新的方法来计算重叠逆转录酶引发位点的SHAPE反应性。
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.
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影响因子：
6.8
作者：
Lu Z;Chang HY
通讯作者：
Chang HY
影响因子：
12.3
作者：
Langmead B;Schatz MC;Lin J;Pop M;Salzberg SL
通讯作者：
Salzberg SL
影响因子：
2.7
作者：
Adams, MJ;Antoniw, JF
通讯作者：
Antoniw, JF
DOI：
10.1073/pnas.1219988110
发表时间：
2013-04-02
影响因子：
11.1
作者：
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通讯作者：
Weeks, Kevin M.
影响因子：
5.8
作者：
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通讯作者：
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