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

丝状病毒（埃博拉病毒[EBOV]和马尔堡病毒[MARV]）和沙状病毒（如拉沙病毒；LAFV）是人畜共患的新出现病原体，可在人类和非人类灵长类动物中引起严重出血热暴发。对病毒-宿主界面的基本理解对于制定未来的治疗干预策略和对策至关重要。线状病毒VP40和LAFV Z基质蛋白部分通过劫持含有ww结构域的特异性宿主蛋白来驱动病毒粒子的组装和出口，这些蛋白可以与其高度保守的PPxY l结构域基序相互作用，这些相互作用代表了抑制这些出血热病毒的出口和传播的新治疗靶点。我们的早期研究鉴定了调节丝状病毒和沙粒病毒出芽的宿主ww结构域蛋白，发现E3泛素连接酶Nedd4、ITCH和WWP1是病毒ppxy介导的出芽的正调节因子。然而，在最近的研究中，我们发现含有WW结构域的蛋白YAP/TAZ、BAG3和WWOX （WW结构域含有氧化还原酶，一种多功能肿瘤抑制因子）是ppxy介导的VP40和Z VLPs表达的负调控因子。YAP/TAZ、BAG3和WWOX作为病毒ppxy介导的出芽的负调节因子的鉴定特别有趣，因为所有这三种蛋白都与血管运动素（Amot）相互作用，Amot是一种含有多ppxy的蛋白，其功能是Hippo通路（YAP/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“主调节因子”。此外，Amot本身的表达和稳定性受PPxY/WW-domain与E3泛素连接酶Nedd4家族相互作用的调控。因此，我们假设Amot是连接病毒输出的正负ww结构域调控因子的中心相互作用因子，并且病毒和宿主PPxY基序之间的模块化模仿以及它们与同一宿主ww结构域蛋白结合的竞争性将对细胞过程和病毒复制和发病机制产生重大影响。事实上，我们首次报道了内源性Amot的表达正调控ppxy介导的EBOV和MARV VP40 VLPs的表达，以及活的EBOV和MARV在细胞培养中的表达和传播。在本研究中，我们将基于我们的新发现，即含PPxY的Amot可以积极调节含PPxY的病毒（包括EBOV和MARV）的出口和传播，并确定VP40/Z - Amot -宿主WW-domain相互作用物之间的竞争性PPxY/WW-domain相互作用是否在体外和体内调节VLPs和活病毒的出口。这些目标的成功完成将提供新的见解，了解这种模块化PPxY/ ww结构域与Amot相互作用的复杂网络如何影响出血热病毒的晚期输出和传播，并为靶向可能代表许多RNA病毒的致命弱点的基本病毒-宿主相互作用的治疗策略提供原理证明。

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.