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

 描述(申请人提供)：节肢动物传播的病毒(虫媒病毒)面临着极端的挑战，因为它们同时适应节肢动物和脊椎动物的宿主。虫媒病毒会急性感染人类和其他多种脊椎动物，但必须持续感染一两种蚊子，才能传播给它们的下一个脊椎动物宿主。由于宿主间的循环，虫媒病毒在小RNA介导的免疫反应和RNA干扰(RNAi)方面遇到了巨大的差异。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。由于脊椎动物和蚊子的RNAi性质不同，虫媒病毒逃避和颠覆这些反应的策略应该根据宿主的不同而同样不同。因此，我们假设虫媒病毒：小RNA的相互作用在蚊子和哺乳动物的宿主之间会有根本的不同。作为一种模型虫媒病毒，我们计划研究基孔肯雅病毒(CHIKV)，它会导致一种痛苦、虚弱的疾病，目前还没有疫苗或治疗方法。这项建议的主要目标是了解CHIKV与不同宿主RNAi组件和响应的相互作用策略。这项工作旨在促进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达内尔实验室合作，将ArgAerte(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蛋白是所有RNAi途径所必需的，并由小RNA引导与mRNA或病毒RNA靶标结合。使用AGO-CLIP，我们可以物理地将小RNA介导的AGO结合映射到病毒基因组。这项技术提供了一个独特的机会来检查寄主物种的小RNA与虫媒病毒相互作用的功能差异。在我们的特定目标中，我们建议使用AGO-CLIP在哺乳动物和蚊子模型中1)体外和2)体内将小RNA介导的AGO与CHIKV基因组结合，并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：宿主的相互作用的了解是有限的，特别是在蚊子媒介中，并且一直是发展抗病毒控制策略的主要障碍。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，并可能为阻断虫媒病毒传播的策略提供依据。在全球层面上，系统绘制虫媒病毒的小RNA图谱将有助于深入了解病毒与不同宿主的抗病毒防御之间的相互作用。

 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.