Translation-Independent Roles of RNA Secondary Structures within the Replication Protein Coding Region of Turnip Crinkle Virus
Translation-Independent Roles of RNA Secondary Structures within the Replication Protein Coding Region of Turnip Crinkle Virus
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DOI：
10.3390/v12030350
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发表时间：
2020-03-01
期刊：
影响因子：
4.7
通讯作者：
Qu, Feng
中科院分区：
文献类型：
--
作者：
Sun, Rong;Zhang, Shaoyan;Qu, Feng
RNA二级结构在正意义（+）RNA病毒感染中发挥着多种作用，但那些位于复制蛋白编码序列上的二级结构很难研究。调节复制蛋白翻译的结构带来了特殊的挑战，因为它们在翻译后步骤中的潜在参与不容易独立于它们在调节翻译中的作用而被识别。在目前的研究中，我们试图通过提供反式病毒复制蛋白来克服这些困难。具体来说，我们将侵染植物的萝卜皱缩病毒（TCV）修饰为不能翻译一种（p88）或两种（p28和p88）复制蛋白的变体，并使用由单独的非复制构建体产生的相应复制蛋白来补充它们的复制。这种方法使我们能够重新检查p28/p88编码区，以寻找TCV复制所需的潜在RNA元件。我们发现，虽然超过三分之一的p88编码序列可以在不显著影响病毒RNA水平的情况下被删除，但两个相对较小的区域，即RSE和IRE，对TCV基因组RNA的强大积累至关重要，而不是亚基因组RNA。特别是，先前发现的调控p28终止密码子的翻译读通以产生p88所需的RSE元件，包含有效复制TCV基因组所需的亚元件。这种新方法在其他病毒中的应用可能揭示对病毒增殖至关重要的新的RNA二级结构。
RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.