Investigating the Formation and Function of Subgenomic Flavivirus RNAs During Flavivirus Infection of the Mosquito Vector

项目总结： 在被节肢动物传播的黄病毒，如登革热(DV)和寨卡病毒(ZV)感染期间，被感染的细胞积累 非编码RNA称为亚基因组黄病毒RNA(SfRNAs)，由病毒基因组rna的3组成 未翻译区域(UTR)。当宿主5到3外切核酸酶Xrn1遇到时产生sfRNA 基因组的3个非编码区中的抗核糖核酸酶核糖核酸(XrRNAs)结构使其停止前进。分析 黄病毒基因组显示，大多数蚊媒传播的黄病毒都有多个xrRNA。 串列“。假设这些结构在功能上是多余的，并且独立地工作于 产生sfRNA。然而，我们实验室的数据显示，在某些黄病毒中， 一个xrRNA被另一个感知，影响其功能。我们假设这种“耦合”或“协调”是 由于串联XrRNA通过中间序列和/或结构相互作用。为了检验这一假设，我们 开发了一种替代报告系统，允许我们测试介入突变的功能影响 登革病毒串联xrRNAs序列。具体地说，我们的化验报告了 产生了sfRNAs，这已经被很好地描述为野生型序列。对于目标1，我们将使用以下内容 系统，以探索多种黄病毒中xrRNA之间的这种偶联机制。此外，这一点 研究还将包括揭示sfRNA干扰蚊子免疫的机制。 回应。具体地说，已经证明sfRNAs能够与哺乳动物DICER相互作用 蛋白质，减少体外形成的小干扰RNA(SiRNA)的数量。由于蚊子依赖于RNA 干扰(RNAi)是它们抵御病毒感染的主要防御措施，哺乳动物和蚊子的Disher蛋白 ，我们假设sfRNAs通过与蚊子中的RNAi反应相互作用来抑制RNAi反应 蚊子RNAi途径的DICER-2(Dcr2)蛋白。在目标2下，我们将利用一系列体外培养的 生化分析以测试sfRNA和Dcr2之间的相互作用是如何发生的，并阐明如何 SfRNA的作用是抑制蚊子媒介的免疫反应。揭开这些主机的动态 病原体的相互作用可能导致减少蚊子sfRNA产生的新策略，以遏制 黄病毒的传播。

Project Summary: During infection by arthropod borne flaviviruses such as Dengue (DV) and Zika (ZV), infected cells accumulate non-coding RNAs termed Subgenomic Flaviviral RNAs (sfRNAs) that consist of the viral genomic RNA’s 3 untranslated region (UTR). sfRNAs are generated when the host 5 to 3 exoribonuclease Xrn1 encounters exoribonuclease-resistant RNA (xrRNAs) structures in the genome’s 3 UTR that halt its progress. Analysis of flaviviral genomes has revealed that the majority of mosquito-borne flaviviruses have multiple xrRNAs “in tandem”. It was hypothesized that these structures were functionally redundant and working independently to generate sfRNA. However, data in our lab has revealed that within certain flaviviruses the structural integrity of one xrRNA is sensed by the other, affecting its function. We hypothesize that this “coupling” or “coordination” is due to tandem xrRNAs interacting through intervening sequences and/or structures. To test this hypothesis, we developed a surrogate reporter system that allows us to test the functional effects of mutations in the intervening sequence of the Dengue virus tandem xrRNAs. Specifically, our assay reports on changes in the patterns of produced sfRNAs, which have been well characterized for the wild type sequences. For aim 1 we will use this system to explore the mechanism of this coupling between xrRNAs in multiple flaviviruses. In addition, this research will also include uncovering the mechanism in which sfRNAs interfere with the mosquito immune response. Specifically, it has been shown that sfRNAs are capable of interacting with the mammalian Dicer protein, reducing the amount of small interfering RNA (siRNA) formed in vitro. Since mosquitos rely on RNA interference (RNAi) as their primary defense against viral infection, and mammalian and mosquito Dicer proteins are well conserved, we hypothesize that sfRNAs dampen the RNAi response in mosquitos by interacting with the Dicer-2 (Dcr2) protein of the mosquito RNAi pathway. Under aim 2 we will utilize a series of in vitro biochemical assays to test how this interaction between sfRNA and Dcr2 is taking place and elucidate on how sfRNAs work to dampen the immune response of the mosquito vector. Uncovering the dynamics of these host pathogen interactions could lead to new strategies for attenuating sfRNA production in mosquitos to curb the spread of flaviviruses.