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])和阿雷纳病毒(例如拉萨病毒；拉萨病毒)是人畜共患病的新兴病原体，会在人类和非人类灵长类动物中引起严重出血热的暴发。对病毒-宿主界面的基本了解对于制定未来的治疗干预战略和对策至关重要。由于丝状病毒VP40和LAFV Z基质蛋白通过劫持含有WW结构域的特定宿主蛋白来驱动病毒粒子的组装和外出，这些宿主蛋白可以与其高度保守的PPxY L结构域相互作用，这些相互作用代表了一个新的治疗靶点，以抑制这些出血热病毒的外出和传播。我们早期对调节丝状病毒和花生病毒萌发的宿主WW结构域蛋白的研究发现，E3泛素连接酶Nedd4、itch和WWP1是病毒PPxY介导的萌发的阳性调节因子。然而，在最近的研究中，我们发现包含WW结构域的蛋白YAP/TAZ、BAG3和现在的WWOX(WW结构域包含氧化还原酶；一个多功能的肿瘤抑制因子)是PPxY介导的VP40和Z VLP出口的负调控因子。YAP/TAZ、BAG3和WWOX是病毒PPxY介导的萌发的负调控因子，这三种蛋白都与血管蛋白(Amot)相互作用，Angiomotin(AMOT)是一种含有多PPxY的蛋白，功能是河马途径(YAP/TAZ)信号、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”，因此鉴定YAP/TAZ、BAG3和WWOX是特别有趣的。此外，AMOT本身的表达和稳定性受到PPxY/WW结构域与E3泛素连接酶Nedd4家族相互作用的调节。因此，我们假设AMOT是连接含有病毒出口调节因子的正负WW结构域的中心相互作用者，并且病毒和宿主PPxY基序之间的模块化模仿以及它们与含有相同宿主WW结构域的蛋白结合的竞争性将对细胞过程和病毒复制和致病产生重要影响。事实上，我们首次报道了内源性AMOT的表达正向调节PPxY介导的EBOV和MARV VP40 VLP的出口，以及在细胞培养中活的EBOV和MARV的出口和传播。在这项提案中，我们将基于我们的新发现，即含有PPxY的AMOT可以正向调节含PPxY的病毒(包括EBOV和MARV)的出口和传播，并确定VP40/Z-AMOT-宿主WW结构域之间的竞争性PPxY/WW结构域相互作用是否在体外和体内调节VLP和活病毒的出口。这些目标的成功实现将为这种与AMOT的模块化PPxY/WW结构域相互作用的复杂网络如何影响出血热病毒出口和传播的晚期阶段提供新的见解，并将作为针对关键病毒-宿主相互作用的治疗策略的原则证明，而这种相互作用可能是许多RNA病毒的致命弱点。

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.