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

寨卡病毒（ZIKV）是一种致畸性人类病原体，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学，包括中央凹的丧失 反射和黄斑色素斑、脉络膜视网膜瘢痕和黄斑萎缩。ZIKV已成为地方病 和美国的本地传播先前已经报道过。结构损坏的长期影响 对视力的影响，以及先天性ZIKV眼病的致病过程开始被理解。 控制正常眼睛发育的信号通路在ZIKV感染期间失调， 没有很好地描述。我们最近通过建立ZIKV感染性眼部免疫系统进行了一系列实验。 细胞培养系统和小鼠模型，以了解结构和分子扰动。为成功 病毒已经进化出各种策略来逃避先天免疫应答以及增强免疫应答。 满足病毒基因组合成的高能量需求所需的细胞代谢物的可用性。 我们发现AMPKα是一种细胞主能量传感器，在ZIKV感染的视网膜细胞中被激活。 此外，AMPK的药理学活化导致ZIKV复制减弱。另一个有趣 发现肿瘤抑制因子Hippo/SWH信号通路中的雅普/TAZ因子被早期诱导 在RPE细胞中，在ZIKV感染的后期降解。沉默雅普/TAZ导致ZIKV降低 复制的由于能量传感器AMPK和Hippo信号通路控制着关键的细胞过程， 包括宿主的抗病毒反应，理解这两种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，以1） 增加细胞内代谢资源，和2）抑制TBK 1以拮抗抗病毒防御。这些分子 这些变化可以通过病毒编码因子协调，导致眼细胞损伤的发病机制。的 将研究以下具体目标。目标1侧重于系统地评估AMPK的作用， Hippo信号传导调节RPE细胞中对ZIKV感染的抗病毒应答。之间的串扰 这些途径将在雅普/TAZ水平上进行研究。药理学激活/抑制和基因 将进行RPE细胞中的敲除方法。目的2被设计为阐明ZIKV对细胞凋亡的影响。 视网膜发育过程中的Hippo和AMPK信号通路。人iPSC衍生的3D视网膜杯 类器官将用于研究视网膜发育和ZIKV介导的视网膜发育失调之间的联系。 这些关键路径。将表征调节这些途径的ZIKV编码的毒力因子。目的 3是确定AMPK、TBK 1和Hippo信号传导的RPE特异性消融对细胞凋亡的影响。 ZIKV诱导的小鼠脉络膜视网膜萎缩的发病机制。这项拟议中的研究将产生新的见解 ZIKV在眼部疾病中的发病机制和潜在治疗靶点的鉴定。

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.