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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 登革热是人类最常见的病媒传播的病毒性疾病。由于缺乏疫苗和药物，登革热的出现和死灰复燃已成为热带和亚热带地区日益严重的威胁人类健康的问题。登革病毒（DENV）是一种正义单链RNA病毒，通过感染的伊蚊（包括Ae. aegypti和Ae.白纹伊蚊了解登革病毒与蚊媒之间的分子相互作用是制定有效的病媒控制策略的先决条件。然而，人们对蚊子如何科普病毒感染以及对病毒的先天免疫是什么知之甚少。 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是保守的。在植物和果蝇中，RNAi已被确定为天然的抗病毒免疫。病毒既是RNAi的启动子又是RNAi的靶点，复制的病毒RNA被RNAi机制识别并加工成病毒小干扰RNA（siRNA），其指导同源病毒RNA的特异性降解。另一方面，病毒编码蛋白质来抑制基于RNAi的抗病毒防御，以实现成功感染。在伊蚊中，靶向DENV序列的工程RNAi可能导致对DENV感染的抗性或抑制病毒复制和积累。但目前尚不清楚RNAi机制是否是蚊子的主要天然抗病毒免疫机制，以及病毒如何与RNAi途径相互作用。 该项目的目的是研究RNAi在伊蚊细胞中响应DENV感染的自然作用。 具体目标1。 登革病毒感染伊蚊细胞中小分子RNA的特征 在植物和果蝇中，病毒感染可以诱导基于RNAi的抗病毒防御，这与来自沉默的病毒基因组的丰富siRNA的积累相关。病毒siRNA被认为是感染细胞中抗病毒RNAi的分子标记。 (1)确定DENV病毒siRNA是否在感染的伊蚊细胞中积累 (2)对来自DENV感染的伊蚊细胞的小RNA进行超深度测序 (3)DENV感染的伊蚊细胞中小RNA的鉴定和表征 具体目标2。 DENV编码的RNAi抑制子的鉴定 除了在感染细胞中检测到病毒siRNA外，DENV编码的RNAi抑制子的鉴定是支持RNAi在伊蚊细胞中作为天然抗病毒免疫的另一个直接证据。 (1)确定DENV感染是否可以抑制伊蚊细胞中的RNAi (2)鉴定可抑制伊蚊细胞中RNAi的DENV蛋白 (3)确定DENV蛋白是否可以抑制哺乳动物细胞中的RNAi (4)通过DENV蛋白进行RNAi抑制的机制分析

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, its 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