UH1 COBRE PILOT: ROLE OF DNA INTERFERENCE IN DENGUE VIRUS-MOSQUITO INTERACTIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 登革热是人类最常见的媒介传播病毒疾病。由于缺乏疫苗和药物，登革热的出现和死灰复燃对热带和亚热带地区的人类健康构成了越来越大的威胁。登革病毒(DENV)是一种阳性的单链RNA病毒，通过包括伊蚊在内的受感染伊蚊的叮咬传播给人类。Aegypti和Ae.白纹伊蚊。了解登革热病毒和媒介蚊子之间的分子相互作用是制定有效的媒介控制策略的先决条件。然而，人们对蚊子如何应对病毒感染以及对病毒的先天免疫力知之甚少。 RNA干扰(RNAi)是一种小RNA介导的基因调控机制，在大多数真核生物中都是保守的。在植物和果蝇中，RNAi已被确定为一种天然的抗病毒免疫。病毒既是RNAi的发起者，也是RNAi的靶标，复制的病毒RNA被RNAi机制识别并加工成病毒小干扰RNA(SiRNAs)，指导同源病毒RNA的特异性降解。另一方面，病毒通过编码蛋白质来抑制基于RNAi的抗病毒防御，从而实现成功感染。在伊蚊中，针对DENV序列进行RNAi工程可能会导致对DENV感染的抵抗力或抑制病毒的复制和积累。然而，在蚊子中，RNAi机制是否作为一种主要的天然抗病毒免疫作用，以及病毒如何与RNAi途径相互作用仍不清楚。 该项目的目的是研究RNAi在蚊子细胞对DENV感染的反应中的自然作用。 特定目的1.DENV感染的伊蚊细胞中小RNA的特性 在植物和果蝇中，病毒感染可以诱导基于RNAi的抗病毒防御，这与沉默的病毒基因组中丰富的siRNAs积累有关。病毒siRNAs被认为是感染细胞中抗病毒RNAi的分子标志。 (1)确定DENV病毒siRNA是否在受感染的伊蚊细胞中积聚 (2)对感染登革热病毒的伊蚊细胞中的小RNA进行超深测序 (3)鉴定和鉴定DENV感染的伊蚊细胞中的小RNA 特定目的2.DENV编码的RNAi抑制子的鉴定 除了检测感染细胞中的病毒siRNAs外，识别DENV编码的RNAi抑制子是支持RNAi在蚊子细胞中发挥天然抗病毒免疫作用的另一个直接证据。 (1)确定DENV感染是否能抑制伊蚊细胞中的RNAi (2)在蚊虫细胞中鉴定能抑制RNAi的DENV蛋白 (3)确定DENV蛋白是否能抑制哺乳动物细胞中的RNAi (4)DENV蛋白抑制RNAi的机理分析

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Dengue is the most common vector-borne viral disease in humans. Because of the lack of vaccines and drugs, emerging and resurging of dengue have become increasing threats to human health in tropical and subtropical regions. Dengue virus (DENV), a positive-sense, single-stranded RNA virus, is transmitted to humans through the bite of infected Aedes mosquitoes, including Ae. aegypti and Ae. albopictus. Understanding the molecular interactions between dengue virus and the vector mosquito is the prerequisite for developing effective strategies for vector control. However, little is known about how mosquitoes cope with virus infection and what the innate immunity against viruses is. RNA interference (RNAi) is a small RNA-mediated gene-regulatory mechanism that is conserved in most eukaryotic organisms. In plants and Drosophila, RNAi has been identified as a natural antiviral immunity. Viruses are both initiators and targets of RNAi, and replicating viral RNAs are recognized by RNAi machinery and processed into viral small interfering RNAs (siRNAs), which direct specific degradation of homologous viral RNAs. On the other hand, viruses encode proteins to suppress RNAi-based antiviral defense to achieve successful infection. In Aedes mosquitoes, engineering RNAi that targets DENV sequences could lead to resistance to DENV infection or inhibition of virus replication and accumulation. However, it's still not clear in mosquitoes if RNAi mechanism functions as a major natural antiviral immunity, and how viruses interact with RNAi pathway. The objective of this project is to investigate the natural role of RNAi in Aedes mosquito cells in response to DENV infection. Specific Aim 1. Characterization of small RNAs in DENV-infected Aedes mosquito cells In plants and Drosophila, RNAi-based antiviral defense can be induced by virus infection, which is correlated with accumulation of abundant siRNAs derived from the silenced viral genome. Viral siRNAs are considered as a molecular signature of the antiviral RNAi in infected cells. (1) Determine if DENV viral siRNAs accumulate in infected Aedes mosquito cells (2) Perform ultra-deep sequencing of small RNAs from DENV-infected Aedes mosquito cells (3) Identify and characterize small RNAs in DENV-infected Aedes mosquito cells Specific Aim 2. Identification of RNAi suppressors encoded by DENV Besides the detection of viral siRNAs in infected cells, identification of RNAi suppressors encoded by DENV is another direct evidence to support that RNAi serves as natural antiviral immunity in Aedes mosquito cells. (1) Determine if DENV infection can inhibit RNAi in Aedes mosquito cells (2) Identify DENV proteins that can inhibit RNAi in Aedes mosquito cells (3) Determine if DENV proteins can suppress RNAi in mammalian cells (4) Perform mechanistic analysis of RNAi suppression by DENV proteins