Elucidating the diverse roles of noncoding small RNAs in chikungunya virus infection

阐明非编码小RNA在基孔肯雅病毒感染中的多种作用

• 批准号: 8983335
• 负责人: Kathryn Rozen-Gagnon
• 金额: $ 5.07万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-11-15 至 2018-11-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8983335
• 关键词: Aedes; Americas; Antibodies; Antiviral Agents; Antiviral Response; Arbovirus Infections; Arboviruses; Arthropods; Attention; Binding; Biology; C57BL/6 Mouse; Cell Culture Techniques; Cell Line; Cells; Cellular Tropism; Chikungunya virus; Culicidae; Data; Development; Drosophila genus; Drug Targeting; Face; Genetic Transcription; Goals; Host Defense; Human; Immune; Immune response; Immunity; Immunoprecipitation; In Vitro; Individual; Infection; Insecta; Invertebrates; Lead; Mammals; Maps; Mediating; Messenger RNA; Methods; MicroRNAs; Modeling; Nature; Pain; Pathway interactions; Population; Predisposition; Prevention strategy; Proteins; Public Health; RNA; RNA Binding; RNA Interference; RNA Interference Pathway; RNA Viruses; Role; Small RNA; Techniques; Tropism; Universities; Vaccines; Vertebrates; Viral; Viral Genome; Virus; Virus Diseases; Western Blotting; Work; cell type; cross reactivity; crosslink; immune RNA; in vivo; in vivo Model; insight; novel; public health relevance; research study; response; tool; transcriptome sequencing; transmission process; vector; vector mosquito; viral RNA; virus host interaction

 DESCRIPTION (provided by applicant): Arthropod-borne viruses (arboviruses) face extreme challenges as they adapt to both arthropod and vertebrate hosts. Arboviruses acutely infect humans and a wide range of other vertebrates, but must persistently infect one or two mosquito species for transmission to their next vertebrate host. Due to interhost cycling, arboviruses encounter vast differences in immune responses and RNA interference (RNAi), mediated by small RNAs. In contrast to in vertebrates, RNAi is the main antiviral response in insects, targeting RNA virus genomes for degradation to limit viral replication. Because of the disparate nature of RNAi in vertebrates versus mosquitoes, strategies for arboviruses to evade and subvert these responses should be equally divergent depending on the host. Therefore, we hypothesize that arbovirus:small RNA interplay will vary radically between mosquito and mammalian hosts. As a model arbovirus, we plan to study chikungunya virus (CHIKV), which causes a painful, debilitating illness, for which there is no vaccine or treatment. The main goal o this proposal is to understand CHIKV interaction strategies with disparate host RNAi components and responses. This work aims to advance our understanding of host-specific small RNA interactions with arboviruses, beginning with CHIKV. Recently, we collaborated with the Darnell lab at The Rockefeller University to apply cross-linking and immunoprecipitation (CLIP) of Argonaute (Ago; termed Ago-CLIP) to virus-infected cells. Ago proteins are essential to all RNAi pathways, and are guided by small RNAs to bind to mRNA or viral RNA targets. Using Ago-CLIP, we can physically map small RNA-mediated Ago binding to viral genomes. This technique offers a unique opportunity to examine functional differences in host species' small RNA interactions with arboviruses. In our specific aims, we propose to use Ago-CLIP to map small RNA-mediated Ago binding to the CHIKV genome in both mammalian and mosquito models 1) in vitro and 2) in vivo, with special attention to interactions relevant to mosquito antiviral responses. Our understanding of arbovirus:host interactions is limited, particularly in mosquito vectors, and has been a major impediment to the development of antiviral control strategies. Our aims will allow us to identify critical CHIKV:small RNA interactions, which may uncover novel drug targets. Furthermore, observation of such small RNA interactions will increase our understanding of determinants of tropism and may inform strategies for disrupting arbovirus transmission. On a global level, systemic mapping of small RNA landscapes for arboviruses will yield insights into the interplay between viruses and antiviral defenses in disparate hosts.

 描述（由申请人提供）：节肢动物传播的病毒（虫媒病毒）在适应节肢动物和脊椎动物宿主时面临着极端的挑战。虫媒病毒可急性感染人类和多种其他脊椎动物，但必须持续感染一种或两种蚊子才能传播到下一个脊椎动物宿主。由于宿主间循环，虫媒病毒在免疫反应和由小 RNA 介导的 RNA 干扰 (RNAi) 方面遇到巨大差异。与脊椎动物不同，RNAi 是昆虫的主要抗病毒反应，针对 RNA 病毒基因组进行降解，以限制病毒复制。由于脊椎动物与蚊子的 RNAi 性质不同，虫媒病毒逃避和破坏这些反应的策略也应根据宿主的不同而有所不同。因此，我们假设蚊子和哺乳动物宿主之间的虫媒病毒：小RNA相互作用将存在根本差异。作为一种虫媒病毒模型，我们计划研究基孔肯雅病毒（CHIKV），这种病毒会导致一种痛苦的、使人衰弱的疾病，而且目前还没有疫苗或治疗方法。该提案的主要目标是了解 CHIKV 与不同宿主 RNAi 成分和反应的相互作用策略。这项工作旨在加深我们对宿主特异性小 RNA 与虫媒病毒相互作用的理解，从 CHIKV 开始。 最近，我们与洛克菲勒大学的 Darnell 实验室合作，将 Argonaute (Ago；称为 Ago-CLIP) 的交联和免疫沉淀 (CLIP) 应用于病毒感染的细胞。 Ago 蛋白对于所有 RNAi 途径至关重要，并由小 RNA 引导与 mRNA 或病毒 RNA 靶标结合。使用 Ago-CLIP，我们可以物理绘制小 RNA 介导的 Ago 与病毒基因组的结合图谱。这项技术提供了一个独特的机会来检查宿主物种小 RNA 与虫媒病毒相互作用的功能差异。在我们的具体目标中，我们建议使用 Ago-CLIP 在哺乳动物和蚊子模型 1) 体外和 2) 体内绘制小 RNA 介导的 Ago 与 CHIKV 基因组的结合图谱，特别关注与蚊子抗病毒反应相关的相互作用。我们对虫媒病毒与宿主相互作用的了解是有限的，特别是在蚊子媒介中，并且一直是抗病毒控制策略发展的主要障碍。我们的目标将使我们能够识别关键的 CHIKV：小 RNA 相互作用，这可能会发现新的药物靶点。此外，对这种小 RNA 相互作用的观察将增加我们对趋向性决定因素的理解，并可能为破坏虫媒病毒传播的策略提供信息。在全球层面上，虫媒病毒小 RNA 景观的系统图谱将深入了解不同宿主中病毒和抗病毒防御之间的相互作用。