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

研究蚊子载体黄病毒感染过程中亚基因组黄病毒 RNA 的形成和功能

• 批准号: 10677398
• 负责人: Elizabeth Spear
• 金额: $ 3.65万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677398
• 关键词: 3' Untranslated Regions; Affect; Arbovirus Infections; Attenuated; Binding; Binding Sites; Biochemical; Biological Assay; Cells; Coupled; Coupling; Culicidae; Data; Dengue; Dengue Infection; Dengue Virus; Double-Stranded RNA; Down-Regulation; Elements; Exoribonucleases; Flavivirus; Flavivirus Infections; Genome; Goals; Human; Immune; Immune Evasion; Immune response; Immunity; In Vitro; Infection; Interferons; Introns; Knowledge; Lead; Maps; Methods; Mutation; Pathogenicity; Pattern; Persons; Production; Proteins; RNA; RNA Interference; RNA Interference Pathway; RNA Viruses; Recombinants; Reporter; Reporting; Repression; Research; Resistance; Ribonucleases; Series; Small Interfering RNA; Small RNA; Structure; System; Testing; Untranslated RNA; Untranslated Regions; Viral Physiology; Virus; Virus Diseases; West Nile virus; Western Blotting; Work; ZIKA; Zika Virus; arm; arthropod-borne; enzyme substrate; genomic RNA; member; mosquito-borne; novel therapeutics; pathogen; prevent; response; spleen exonuclease; stem; vector; vector mosquito; viral fitness; viral genomics

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.

项目概要： 在被节肢动物传播的黄病毒如登革热（DV）和寨卡（ZV）感染期间， 称为亚基因组黄病毒RNA（sfRNA）的非编码RNA，由病毒基因组RNA的3 '组成， 非翻译区（UTR）。当宿主5 - 3 bp的核糖核酸外切酶Xrn 1遇到 在基因组的3个非编码区中，核糖核酸外切酶抗性RNA（xrRNA）结构阻止了其进展。分析 黄病毒基因组显示，大多数蚊媒黄病毒在体内都有多个xrRNA“。 串联”。据推测，这些结构在功能上是冗余的，并且独立工作， 生成sfRNA。然而，我们实验室的数据显示，在某些黄病毒中， 一种xrRNA被另一种xrRNA感知，从而影响其功能。我们假设这种“耦合”或“协调”是 由于串联xrRNA通过插入序列和/或结构相互作用。为了验证这个假设，我们 开发了一个替代报告系统，使我们能够测试突变的功能影响，在干预 登革病毒串联xrRNA的序列。具体地说，我们的分析报告了 产生了sfRNA，其已被很好地表征为野生型序列。对于目标1，我们将使用 系统来探索多种黄病毒中xrRNA之间的这种偶联机制。另外这款 研究还将包括揭示sfRNA干扰蚊子免疫的机制， 反应具体地，已经显示sfRNA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）的量。因为蚊子依赖RNA 干扰（RNAi）作为它们对抗病毒感染的主要防御，以及哺乳动物和蚊子Dicer蛋白 是很保守的，我们假设sfRNA通过与蚊子中的RNA干扰分子相互作用来抑制蚊子中的RNAi反应。 蚊子RNAi途径的Dicer-2（Dcr 2）蛋白。在目标2下，我们将利用一系列体外 生物化学测定来测试sfRNA和Dcr 2之间的这种相互作用是如何发生的，并阐明sfRNA和Dcr 2之间的相互作用如何发生。 sfRNA用于抑制蚊子载体的免疫应答。揭示这些宿主的动态 病原体的相互作用可能会导致新的策略，减少蚊子中sfRNA的产生，以抑制蚊子的免疫反应。 黄病毒的传播。