Interplay between AMPK and Hippo Signaling Regulates Ocular Antiviral Response to Zika virus infection

项目摘要：寨卡病毒(ZIKV)是一种人类致畸病原体，可导致先天性眼睛和大脑 疾病。受影响的婴儿表现出视力障碍和相关的眼部病理，包括中心凹丧失。 反射和黄斑色素斑驳，脉络膜视网膜疤痕和黄斑萎缩。寨卡病毒已经成为地方病 此前，美国也有过当地传播病毒的报道。结构损伤的长期影响 对视力的影响，以及先天性ZIKV眼病的致病过程开始被理解。 在ZIKV感染过程中，调控正常眼睛发育的信号通路是 不是很好的特征。我们最近进行了一系列实验，建立了ZIKV感染性眼球 细胞培养系统和小鼠模型，以了解结构和分子的扰动。为了成功 在复制过程中，病毒已经进化出各种策略来逃避先天免疫反应以及增强 满足病毒基因组合成所需的高能量需求所需的细胞代谢物的可用性。 我们发现，在感染ZIKV的视网膜细胞中，AMPKα被激活，这是一种细胞主能量感受器。 此外，AMPK的药理激活导致ZIKV复制减弱。另一件有趣的事 研究发现，肿瘤抑制因子Hippo/SWH信号通路中的YAP/TAZ因子被早期诱导 在RPE细胞中，ZIKV感染后期降解。消除YAP/TAZ的沉默导致ZIKV减少 复制。由于能量传感器AMPK和河马信号通路控制着关键的细胞过程， 包括宿主的抗病毒反应在内，了解这两条途径的基本机制是至关重要的 放松管制。我们假设ZIKV调节眼细胞中的AMPK和HIPPO信号通路到1) 增加细胞内代谢资源，以及2)抑制TBK1拮抗抗病毒防御。这些分子 改变可以通过病毒编码的因子来协调，从而导致眼细胞损伤的发病机制。这个 将对以下具体目标进行调查。目的1系统评价AMPK的作用。 河马信号在调节RPE细胞对ZIKV感染的抗病毒反应中的作用。两个人之间的串音 这些途径将在YAP/TAZ一级进行调查。药理激活/抑制和基因 将在RPE细胞中进行基因敲除方法。目的2旨在阐明ZIKV对 河马和AMPK信号通路在视网膜发育中的作用。人IPSC衍生3D-视网膜杯 有机化合物将被用于研究视网膜发育和ZIKV介导的放松调控之间的联系。 这些关键路径。调控这些途径的ZIKV编码的毒力因子将被描述。目标 3是确定RPE特异性消融AMPK、TBK1和河马信号对 ZIKV诱导的小鼠脉络膜视网膜萎缩的发病机制这项拟议的研究将产生新的见解 探讨ZIKV在眼科疾病中的发病机制，并确定潜在的治疗靶点。

PROJECT SUMMARY: Zika virus (ZIKV) is a teratogenic human pathogen that causes congenital eye and brain diseases. Affected babies exhibit vision impairment and associated ocular pathology, including loss of foveal reflex and macular pigment mottling, chorioretinal scarring, and macular atrophy. ZIKV has become endemic and local transmissions in the USA have been reported previously. The long-term effects of structural damage on vision, as well as the pathogenic processes of congenital ZIKV eye diseases are beginning to be understood. The signaling pathways governing normal eye development, which are dysregulated during ZIKV infection, are not well characterized. We recently carried out a series of experiments by establishing a ZIKV infectious ocular cell culture system and mouse models to understand the structural and molecular perturbations. For successful replication, viruses have evolved various strategies to evade innate immune response as well as to enhance the availability of cellular metabolites required to meet the heightened energy demand for viral genome synthesis. We found that the AMPKα, a cellular master energy sensor, is activated in the ZIKV-infected retinal cells. Moreover, pharmacological activation of AMPK resulted in attenuated ZIKV replication. Another interesting finding is that the YAP/TAZ factors in the tumor suppressor Hippo/SWH signaling pathway were induced early on, but degraded at later stage of ZIKV infection in RPE cells. Silencing YAP/TAZ resulted in reduced ZIKV replication. Since the energy sensor AMPK and Hippo signaling pathways control key cellular processes, including host antiviral responses, it is critical to understand the fundamental mechanism of these two pathways deregulation. We hypothesize that ZIKV modulates AMPK and Hippo signaling pathways in ocular cells to 1) increase intracellular metabolic resources, and 2) inhibit TBK1 to antagonize antiviral defense. These molecular changes can be orchestrated through viral coded factors resulting in the pathogenesis of ocular cell injury. The following specific aims will be investigated. Aim 1 focuses on systematically evaluating the role of AMPK- Hippo signaling on regulating antiviral response to ZIKV infection in RPE cells. The cross talk between these pathways will be investigated at the YAP/TAZ level. Pharmacological activation/inhibition, and gene knockout approaches in RPE cells will be carried out. Aim 2 is designed to elucidate the effect of ZIKV on Hippo and AMPK signaling pathways during retinal development. Human iPSC-derived 3D-retinal cup organoids will be used to investigate the link between retinal development and ZIKV-mediated deregulation of these key pathways. The ZIKV-encoded virulence factors regulating these pathways will be characterized. Aim 3 is to determine the effect of RPE-specific ablation of AMPK, TBK1, and Hippo signaling on the pathogenesis of ZIKV-induced chorioretinal atrophy in mice. This proposed study would yield novel insights into the pathogenesis of ZIKV in ocular diseases and identification of potential therapeutic targets.