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

项目总结/摘要 许多病毒使用非编码RNA来操纵宿主细胞过程。一个这样的RNA的例子被发现 在节肢动物传播的黄病毒（FV）中，一类负责严重公共卫生威胁的病毒，包括 登革热、黄热病和西尼罗河病毒。FV产生亚基因组黄病毒RNA（sfRNA），RNA 长度约200-500 nt的片段，已发现其与FV的病理有关 感染模型中的感染系统。sfRNA片段通过全长黄病毒的裂解产生， 通过内源性5 '→ 3'外切核酸酶Xrn 1对基因组进行修饰。sfRNA内的特定结构RNA元件 抵抗Xrn 1降解，停止Xrn 1切割并留下sfRNA片段。这些结构化的RNA 元件被称为Xrn 1抗性RNA（xrRNA），并且发现xrRNA的突变可以消除Xrn 1抗性RNA。 在黄病毒感染期间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。的 RNA的折叠暗示了Xrn 1抗性的潜在机制，然而目前还不清楚是否 RNA形成静态的、高度稳定的RNA结构或通过动态相互作用实现其活性 使用Xrn 1入口通道。对xrRNA的完整机制理解需要理解 xrRNA折叠的稳定性和分子动力学。在我的第一个目标中，我建议使用单分子福斯特 共振能量转移（FRET）结合Xrn 1抗性的定量生化测定， 量化xrRNA中的折叠转变，并证明xrRNA折叠如何有助于Xrn 1抗性。 有几个不同的谱系节肢动物-bourne FV，包括蚊子-bourne（MB），蜱-bourne (TB)、未知载体（NKV）和昆虫特异性黄病毒（ISF）。这些sfRNA序列来源于 谱系高度分化，因此需要进一步研究TB、NKV和ISF谱系sfRNA， 确定它们如何抵抗Xrn 1降解。在我的第二个目标中，我建议用生物化学的方法来描述 来自TB、NKV和ISF谱系的xrRNA元件，以确定sfRNA的哪些区域是 负责Xrn 1抗性并决定其二级结构。我还建议确定三个- 通过X射线晶体学研究这些元素的三维结构。 已经发现sfRNA通过与Xrn 1形成复合物并将其隔离来抑制Xrn 1的活性， 在感染期间改变RNA转录物的调节。在我的第三个目标中，我建议进一步研究这种影响。我 为了确定最小的RNA序列， Xrn 1螯合活性的要求，并确定sfRNA如何与Xrn 1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.