Role of Host Angiomotin as a Central Regulator of Filovirus Egress and Dissemination

丝状病毒（埃博拉[EBOV]和马尔堡[MARV]）和沙粒病毒（例如拉沙病毒; LAFV）是引起人类和非人灵长类动物中严重出血热暴发的人畜共患的新兴病原体。对病毒-宿主界面的基本了解对于制定未来的治疗干预策略和对策至关重要。由于丝状病毒VP 40和LAFV Z基质蛋白部分通过劫持含有可以与其高度保守的PPxY L结构域基序相互作用的WW结构域的特定宿主蛋白来驱动病毒粒子组装和逸出，因此这些相互作用代表了一种新型治疗靶点来抑制这些出血热病毒的逸出和传播。我们的早期研究，以确定宿主的WW-domain蛋白，调节丝状病毒和沙粒病毒出芽确定E3泛素连接酶Nedd 4，ITCH，和WWP 1作为病毒PPxY介导的出芽的正调节剂。然而，在最近的研究中，我们鉴定了含WW结构域的蛋白质雅普/TAZ、BAG 3，以及现在的WWOX（含WW结构域的氧化还原酶;多功能肿瘤抑制剂）作为PPxY介导的VP 40和Z VLP排出的负调节剂。将雅普/TAZ、BAG 3和WWOX鉴定为病毒PPxY介导的出芽的负调节剂是特别有趣的，因为所有这三种蛋白质都与血管动蛋白（Amot）相互作用，血管动蛋白是一种含有多PPxY的蛋白质，其作为Hippo途径（雅普/TAZ）信号传导、细胞骨架动力学、细胞迁移/增殖和紧密连接（TJ）完整性的“主调节剂”发挥作用。此外，Amot本身的表达和稳定性受PPxY/WW-domain与E3泛素连接酶Nedd 4家族相互作用的调节。因此，我们假设Amot是连接病毒外出的正和负的含有WW-domain的调节剂两者的中心相互作用物，并且病毒和宿主PPxY基序之间的模块化模拟以及它们与含有相同宿主WW-domain的蛋白质结合的竞争性质将对细胞过程和病毒复制和发病机制两者产生重大影响。事实上，我们首先报道了内源Amot的表达正调节PPxY介导的EBOV和MARV VP 40 VLP的流出以及活EBOV和MARV在细胞培养物中的流出和扩散。在该提议中，我们将基于我们的新发现，即含PPxY的Amot可以正向调节含PPxY的病毒（包括EBOV和MARV）的外出和扩散，并确定VP 40/Z-Amot-宿主WWW结构域相互作用物之间的竞争性PPxY/WWW结构域相互作用是否在体外和体内调节VLP和活病毒的外出。这些目标的成功完成将提供新的见解，这种复杂的网络模块PPxY/WW-domain与Amot的相互作用如何影响出血热病毒的出口和传播的后期阶段，并作为针对一个重要的病毒-宿主相互作用，可能代表了许多RNA病毒的致命弱点的治疗策略的原则证明。

Filoviruses (Ebola [EBOV] and Marburg [MARV]) and arenaviruses (e.g. Lassa virus; LAFV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans and non-human primates. A fundamental understanding of the virus-host interface is critical for developing future strategies and countermeasures for therapeutic intervention. As the filovirus VP40 and LAFV Z matrix proteins drive virion assembly and egress, in part, by hijacking specific host proteins containing WW-domains that can interact with their highly conserved PPxY L-domain motifs, these interactions represent a novel therapeutic target to inhibit egress and dissemination of these hemorrhagic fever viruses. Our early studies to identify host WW-domain proteins that regulate filovirus and arenavirus budding identified the E3 ubiquitin ligases Nedd4, ITCH, and WWP1 as positive regulators of viral PPxY-mediated budding. However, in more recent studies, we identified WW-domain containing proteins YAP/TAZ, BAG3, and now WWOX (WW Domain Containing Oxidoreductase; a multi-functional tumor suppressor), as negative regulators of PPxY-mediated egress of VP40 and Z VLPs. The identification of YAP/TAZ, BAG3, and WWOX as negative regulators of viral PPxY-mediated budding is particularly intriguing since all three of these proteins interact with Angiomotin (Amot), a multi-PPxY containing protein that functions as a “master regulator” of Hippo pathway (YAP/TAZ) signaling, cytoskeletal dynamics, cell migration/proliferation, and tight junction (TJ) integrity. Moreover, expression and stability of Amot itself are regulated by PPxY/WW-domain interactions with the Nedd4 family of E3 ubiquitin ligases. Thus, we hypothesize that Amot is a central interactor linking both the positive and negative WW-domain containing regulators of virus egress, and that modular mimicry between viral and host PPxY motifs and the competitive nature of their binding to the same host WW-domain containing proteins will have a major impact on both cellular processes and viral replication and pathogenesis. Indeed, we were first to report that expression of endogenous Amot positively regulates PPxY-mediated egress of EBOV and MARV VP40 VLPs as well as egress and spread of live EBOV and MARV in cell culture. In this proposal, we will build upon our novel finding that PPxY-containing Amot can positively regulate egress and spread of PPxY-containing viruses including EBOV and MARV, and determine whether the competitive PPxY/WW-domain interplay among VP40/Z – Amot – host WW-domain interactors regulates egress of VLPs and live viruses in vitro and in vivo. Successful completion of these aims will provide novel insights into how this complex network of modular PPxY/WW-domain interactions with Amot impacts late stages of hemorrhagic fever virus egress and dissemination and serve as proof-of principle for therapeutic strategies targeting an essential viral-host interaction that may represent an Achilles heel for numerous RNA viruses.