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： 在节肢动物传播的黄病毒（例如登革热（DV）和寨卡病毒（ZV））感染期间，受感染的细胞会积累 非编码 RNA 称为亚基因组黄病毒 RNA (sfRNA)，由病毒基因组 RNA 的 3 个组成 当宿主 5 至 3 个核糖核酸外切酶 Xrn1 遇到时，会产生非翻译区 (UTR)。 基因组 3 UTR 中的抗核糖核酸酶 RNA (xrRNA) 结构阻碍了其进展的分析。 黄病毒基因组显示，大多数蚊媒黄病毒“在 串联”是首创的，这些结构在功能上是冗余的并且独立工作。 然而，我们实验室的数据表明，在某些黄病毒中，sfRNA 的结构完整性 一个xrRNA被另一个xrRNA感知，影响其功能，我们发现这种“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。 开发了一种替代报告系统，使我们能够测试干预中突变的功能影响 具体来说，我们报告了检测模式的变化。 产生的 sfRNA 已被很好地表征为野生型序列，我们将使用它来实现目标 1。 系统探索多种黄病毒中 xrRNA 之间的偶联机制。 研究还将包括揭示 sfRNA 干扰蚊子免疫的机制 具体来说，已经证明 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）作为对抗病毒感染的主要防御手段，以及哺乳动物和蚊子的 Dicer 蛋白 非常保守，我们敢说 sfRNA 通过与 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研究。 生化测定来测试 sfRNA 和 Dcr2 之间的相互作用如何发生并阐明如何发生 sfRNA 可以抑制蚊子媒介的免疫反应，揭示这些宿主的动态。 病原体相互作用可能会导致减少蚊子 sfRNA 产生的新策略，以遏制 黄病毒的传播。