Novel innate immune sensing mechanisms of intracellular RNA

项目总结 许多实验室的研究已经证实，DNA传感器--包括膜传感器--定位于 Toll样受体(例如TLR9)和细胞内DNA传感器(例如cGAS和IFI16)-Sense 疱疹病毒感染。与之形成鲜明对比的是，我们对细胞质相关的知识 RIG-I样受体(RLR)家族的RNA传感器在检测疱疹病毒中的残留 难以捉摸。此外，RIG-I和RIG-I识别的生理RNA分子 疱疹病毒感染过程中的相关传感器MDA5目前尚不清楚。 这项拟议的研究建立在Gack实验室最近的一项发现基础上，即RIG-I和 细胞内DNA传感器cGAS和IFI16检测人单纯疱疹病毒1型感染 在时间上截然不同的方式。使用下一代rna测序(rna-seq)，我们鉴定了 结合并激活的是细胞非编码RNA的子集，而不是病毒RNA RIG-I在HSV-1感染过程中的作用。单纯疱疹病毒1型感染者的分子和细胞生物学研究 细胞发现，这些宿主衍生的RNA的自然生命周期的病毒扰动允许 它们被传感器RIG-I识别，从而刺激先天免疫反应。 结合使用一系列协调的分子、生物化学和细胞生物学方法 随着RNA-seq技术在HSV-1感染细胞中的应用，我们将准确地鉴定和表征 RIG-I和MDA5在HSV-1感染过程中识别的生理性RNA配体 (目标1)。此外，我们将定义病毒如何扰乱生命的分子细节 免疫刺激RNA的循环触发RLR介导的免疫反应(目标2)。我们的 研究将提供对RLR介导的疱疹病毒先天感知的分子理解 感染，这可能为新的治疗方法提供基础或帮助设计 更好的疫苗。

PROJECT SUMMARY Work from many laboratories has established that DNA sensors – both membrane-localized Toll-like receptors (e.g. TLR9) and intracellular DNA sensors (e.g. cGAS and IFI16) – sense herpesviral infection. In striking contrast, our knowledge about the relevance of cytoplasmic RNA sensors of the RIG-I-like receptor (RLR) family in the detection of herpesviruses remains elusive. Furthermore, the physiological RNA molecules that are recognized by RIG-I and the related sensor MDA5 during herpesvirus infection are currently unknown. The proposed study builds on a recent discovery by the Gack laboratory that RIG-I and the intracellular DNA sensors cGAS and IFI16 sense herpes simplex virus 1 (HSV-1) infection in a temporally distinct manner. Using next-generation RNA sequencing (RNA-seq), we identified that a subset of cellular non-coding RNAs, rather than viral RNAs, are bound to and activate RIG-I during HSV-1 infection. Molecular and cell biological studies in HSV-1-infected human cells revealed that viral perturbation of the natural life cycle of these host-derived RNAs allows their recognition by the sensor RIG-I, thereby stimulating the innate immune response. Using a coordinated series of molecular, biochemical and cell biological approaches combined with RNA-seq technology in HSV-1-infected cells, we will identify and characterize in precise detail the physiological RNA ligands recognized by RIG-I and MDA5 during HSV-1 infection (Aim 1). Furthermore, we will define the molecular details of how viral perturbation of the life cycle of immunostimulatory RNAs triggers an RLR-mediated immune response (Aim 2). Our studies will provide a molecular understanding of RLR-mediated innate sensing of herpesviral infection, which may provide the foundation for new therapeutic approaches or help design better vaccines.