Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments.
Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments.
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DOI：
10.7554/elife.27453
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发表时间：
2017-09-18
期刊：
影响因子：
7.7
通讯作者：
Lamb DC
中科院分区：
文献类型：
--
作者：
Borodavka A;Dykeman EC;Schrimpf W;Lamb DC
分节RNA病毒是普遍存在的病原体，包括流感病毒和轮状病毒。了解它们的组装的一个主要挑战是一个非随机选择的完整基因组的独特rna的组合问题。这个过程涉及复杂的RNA-RNA和蛋白质- rna相互作用，当浓度接近体内组装条件时，这些相互作用往往被非特异性结合所掩盖。在这里，我们提供了轮状病毒RNA之间序列特异性片段间相互作用的直接实验证据，发生在复杂的RNA和蛋白质丰富的环境中。我们发现轮状病毒编码的非结构蛋白NSP2与病毒ssrna结合导致RNA的重塑，这有利于形成稳定的片段间接触。为了确定这些相互作用的位点，我们开发了一种RNA-RNA SELEX方法来绘制区段间碱基配对所涉及的序列。我们的发现阐明了轮状病毒片段间相互作用的分子基础，为描述控制其他片段RNA病毒组装的类似RNA-RNA相互作用铺平了道路。轮状病毒是一种传染性很强的病毒，影响全世界的儿童，引起严重的腹泻。尽管引进了几种高效疫苗，每年仍有20多万儿童死于轮状病毒。目前还没有药物可以在儿童感染后对抗这种疾病。病毒在DNA或RNA分子中携带决定其性质和行为的指令。与其他通常只有一个DNA或RNA分子的病毒不同，轮状病毒有11个不同的“RNA片段”。病毒侵入细胞后开始自我复制。在复制过程中，RNA片段（由成对的两条RNA链组成）被复制多次。目前尚不清楚轮状病毒如何“计数”到11个，以便每个新病毒获得每个片段的单个副本。先前对轮状病毒复制的生化和结构研究表明，选择11个不同的RNA片段必须涉及RNA与蛋白质和其他RNA分子形成复杂的相互作用。Borodavka等人使用一种高度敏感的基于荧光的方法，称为荧光相互关联光谱，现在提出了通过轮状病毒RNA单链发生的RNA片段之间相互作用的直接实验证据。这些RNA-RNA相互作用需要轮状病毒蛋白NSP2与RNA链结合，从而导致RNA的重塑；这种重塑是在不同RNA片段之间形成稳定接触所必需的。此外，Borodavka等人开发的一种新的实验方法（称为RNA-RNA SELEX）确定了可能参与这些相互作用的RNA片段的部分。Borodavka等人提出的结果为鉴定控制其他分段RNA病毒如何包装其遗传物质的RNA-RNA相互作用铺平了道路。揭示轮状病毒中整个RNA相互作用网络的进一步工作也将加速新疫苗的设计，并可能帮助我们开发治疗感染的抗病毒药物。
Segmented RNA viruses are ubiquitous pathogens, which include influenza viruses and rotaviruses. A major challenge in understanding their assembly is the combinatorial problem of a non-random selection of a full genomic set of distinct RNAs. This process involves complex RNA-RNA and protein-RNA interactions, which are often obscured by non-specific binding at concentrations approaching in vivo assembly conditions. Here, we present direct experimental evidence of sequence-specific inter-segment interactions between rotavirus RNAs, taking place in a complex RNA- and protein-rich milieu. We show that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of stable inter-segment contacts. To identify the sites of these interactions, we have developed an RNA-RNA SELEX approach for mapping the sequences involved in inter-segment base-pairing. Our findings elucidate the molecular basis underlying inter-segment interactions in rotaviruses, paving the way for delineating similar RNA-RNA interactions that govern assembly of other segmented RNA viruses. Rotavirus is a highly infectious virus that affects children worldwide, causing severe diarrhoea. Despite the introduction of several highly effective vaccines, more than 200,000 children still die from rotavirus each year. There are currently no drugs that can combat this disease once a child has been infected. Viruses carry the instructions that determine their properties and behavior in molecules of DNA or RNA. Unlike many other viruses, which typically have a single molecule of DNA or RNA, rotavirus has 11 distinct “RNA segments”. After invading a cell the virus begins to replicate itself. During replication, the RNA segments (which consist of two strands of RNA paired together) are copied many times. It is not clear how rotaviruses ‘count’ up to 11 so that each new virus acquires a single copy of each segment. Previous biochemical and structural studies of rotavirus replication suggest that selecting 11 distinct RNA segments must involve the RNAs forming complex interactions with proteins and other RNA molecules. Using a highly sensitive fluorescence-based approach, termed fluorescence cross-correlation spectroscopy, Borodavka et al. now present direct experimental evidence of interactions between the RNA segments that occur via single strands of the rotavirus RNA. These RNA-RNA interactions require the binding of a rotavirus protein NSP2 to the RNA strands, which results in the remodeling of the RNA; this remodeling is required to form stable contacts between different RNA segments. Furthermore, a new experimental approach (called RNA-RNA SELEX) developed by Borodavka et al. identified the parts of the RNA segments that may take part in these interactions. The results presented by Borodavka et al. pave the way for identifying the RNA-RNA interactions that govern how other segmented RNA viruses can package their genetic material. Further work to uncover the entire RNA interaction network in rotaviruses would also accelerate the design of new vaccines and may help us to develop antiviral drugs to treat infections.