A pathogenic RNA that resists degradation: characterization of subgenomic flavivirus RNA

项目摘要/摘要 许多病毒使用非编码RNA来操纵宿主细胞过程。发现了一个这样的RNA的例子 在节肢动物传播的黄病毒(FV)中，一类对严重公共卫生威胁负责的病毒，包括 登革热、黄热病和西尼罗河病毒。抗体产生亚基因组黄病毒RNA(SfRNAs)，RNA 长度约200-500个核苷酸的片段，已被发现与FV的病理有关 模型感染系统中的感染。SfRNA片段是由全长黄病毒裂解而产生的。 基因组内源5‘→3’外切酶Xrn1。SfRNA中的特定结构RNA元件 抵抗Xrn1的降解，阻止Xrn1的切割，并留下sfRNA片段。这些结构化的RNA 这些元素被称为Xrn1抗性RNAs(XrRNAs)，而xrRNAs的突变被发现可以消除 黄病毒感染过程中sfRNA片段的积累。 Kieft实验室的一项结构研究已经确定了xrRNA元件的三维折叠。这个 RNA的折叠暗示了Xrn1抗性的潜在机制，但目前尚不清楚 RNA形成一种静态的、高度稳定的RNA结构，或通过动态相互作用来完成其活动 具有Xrn1入射通道。对xrRNA的完全机械论理解需要理解 XrRNA折叠的稳定性和分子动力学。在我的第一个目标中，我建议使用单分子Förster 共振能量转移(FRET)结合Xrn1抗性定量生化检测 量化xrRNA中的折叠转变，并展示xrRNA折叠如何有助于Xrn1抗性。 有几种不同的节肢动物-Bourne FV，包括蚊子-Bourne(MB)，嘀嗒-Bourne (TB)、未知媒介(NKV)和昆虫特异性黄病毒(ISF)。从这些序列衍生的sfRNA序列 谱系高度不同，因此需要对TB、NKV和ISF谱系sfRNA进行进一步研究 确定它们如何抵抗Xrn1降解。在我的第二个目标中，我建议从生物化学的角度来描述 来自Tb、NKV和ISF谱系的xrRNA元件，以便确定sfRNA的哪些区域是 负责Xrn1抗性并确定其二级结构。我还建议确定以下三项： 用X射线结晶学研究了这些元素的空间结构。 已经发现sfRNAs通过与Xrn1形成络合物并隔离它来抑制Xrn1的活性， 在感染过程中改变RNA转录本的调控。在我的第三个目标中，我建议进一步研究这种影响。我 已经建立了一种Xrn1抗性的定量测试，以确定最小的RNA序列 对Xrn1封存活动的要求，并确定sfRNAs如何与Xrn1相互作用。

Project Summary/Abstract Many viruses use non-coding RNAs to manipulate host cell processes. An example of one such RNA is found in arthropod-borne flaviviruses (FVs), a class of virus responsible for serious public health threats including Dengue, Yellow Fever, and West Nile Virus. FVs produce subgenomic flavivirus RNAs (sfRNAs), RNA fragments about 200-500 nts in length which have been found to be responsible for the pathology of FV infection in model infection systems. sfRNA fragments are generated by cleavage of the full-length flaviviral genome by the endogenous 5’→3’ exonuclease Xrn1. Specific structured RNA elements within the sfRNA resist Xrn1 degradation, halting Xrn1 cleavage and leaving the sfRNA fragment behind. These structured RNA elements are known as Xrn1-resistant RNAs (xrRNAs), and mutations to xrRNAs are found to eliminate the accumulation of sfRNA fragments during flavivirus infection. A structural study in the Kieft laboratory has identified the three-dimensional fold of an xrRNA element. The folding of the RNA hints at potential mechanisms for Xrn1 resistance, however it is currently unclear whether the RNA forms a static, highly-stable RNA structure or accomplishes its activity through dynamic interactions with the Xrn1 entrance channel. A full mechanistic understanding of xrRNAs requires an understanding of the stability and molecular dynamics of xrRNA folding. In my first aim, I propose to use single-molecule Förster resonance energy transfer (FRET) combined with quantitative biochemical assays of Xrn1 resistance in order to quantify folding transitions in xrRNAs and demonstrate how xrRNA folding contributes to Xrn1 resistance. There are several distinct lineages of arthropod-bourne FVs, including mosquito-bourne (MB), tick-bourne (TB), no known vector (NKV), and insect specific flaviviruses (ISF). The sfRNA sequences derived from these lineages are highly divergent, therefore further study of TB, NKV, and ISF lineage sfRNA is required to determine how they resist Xrn1 degradation. In my second aim, I propose to biochemically characterize the xrRNA elements from TB, NKV, and ISF lineages in order to determine which regions of the sfRNA are responsible for Xrn1 resistance and determine their secondary structure. I also propose to determine the three- dimensional structure of these elements by X-ray crystallography. sfRNAs have been found to inhibit the activity of Xrn1 by forming a complex with Xrn1 and sequestering it, altering the regulation of RNA transcripts during infection. In my third aim I propose to further study this effect. I have created a quantitative Xrn1 resistance assay in order to determine the minimal RNA sequence requirements for Xrn1 sequestration activity and identify how sfRNAs interact with Xrn1.