Regulation of Ebola virus replication by the host ubiquitin system

西非最近爆发的埃博拉病毒(EBOV)感染人数达到令人震惊的水平， 死亡，促使广泛努力制定抗病毒战略；然而，到目前为止还没有得到批准 在人类身上使用。抗病毒药物的开发主要是基于设计靶向 病毒蛋白，但这种方法可能会选择病毒逃逸突变体，并可能发生回复毒力。在……里面 相反，识别病毒复制所需的宿主细胞因子可能会导致新的治疗方法 具有最小固有逃逸突变风险的策略。宿主泛素系统是一个保守的细胞 途径在许多功能中很重要，包括先天免疫信号。众所周知，病毒可以操纵 泛素系统为了自身的优势；因此，本提案的目标是定义分子 涉及泛素系统的EBOV复制机制，并长期应用于此 具有开发有效的抗病毒方法的知识。 EBOV VP35是一种在致病过程中起重要作用的病毒蛋白，因为它是一种重要的辅因子 病毒聚合酶的活性，从而促进有效复制。此外，VP35还能有效地阻断先天免疫 通过抑制宿主的抗病毒I型干扰素(干扰素-I)系统。然而，细胞对VP35功能的调节 翻译后的修饰不被理解。我们的初步数据表明，EBOV复制是 由TRIM6调节VP35泛素化的新机制，一个宿主E3-泛素连接酶，已知 抗病毒功能。根据我们的数据，我们假设VP35劫持TRIM6是为了促进EBOV复制 通过泛素化，同时也阻断了TRIM6的先天免疫信号活性。通过在体外和体内使用 方法，包括我们新产生的TRIM6基因敲除小鼠，我们将测试TRIM6如何在 EBOV复制与致病机制。我们提出了以下具体目标：1)确定机制角色 VP35泛素化在EBOV复制中的作用。我们将使用现有的EBOV VP35病毒突变体，这些病毒突变体缺乏 并测试它们的功能与先天免疫拮抗或病毒聚合酶功能的关系。 2)确定TRIM6调控EBOV复制的分子机制。我们将研究如何 TRIM6通过TRIM6基因敲除细胞株及体外泛素化和结合调节EBOV复制 化验。我们还将证明由TRIM6在体内合成的多Ub链促进EBOV的复制， 使用我们的TRIM6-/-小鼠和小鼠适应的WT EBOV和缺乏泛素化的VP35突变体。 这些研究的结果意义重大，因为它将提供关于 EBOV复制机制，可能适用于其他病毒。因此，一个通用的方法 广谱抗病毒药物的开发可能会被揭示。重要的是，这项工作将告知宿主是否 细胞因子，TRIM6，可能被用作开发新的治疗策略的靶点。 人类最致命的病毒。

The most recent outbreak of Ebola virus (EBOV) in West Africa reached alarming numbers of infections and deaths, prompting extensive efforts to develop antiviral strategies; however, none have thus far been approved for use in humans. The development of antiviral drugs is mostly based on designing compounds that target viral proteins, but this approach may select for viral escape mutants, and reversion to virulence can occur. In contrast, the identification of host cellular factors required for virus replication may lead to novel therapeutic strategies with a minimal inherent risk of escape mutants. The host ubiquitin system is a conserved cellular pathway important in many functions, including innate immune signaling. Viruses are known to manipulate the ubiquitin system for their own advantage; therefore, the goal of this proposal is to define the molecular mechanisms of EBOV replication involving the ubiquitin system with the long-term goal of applying this knowledge in the development of effective antiviral approaches. EBOV VP35 is a critical viral protein contributing to pathogenesis because it serves as an essential cofactor of the viral polymerase, thereby promoting efficient replication. Further, VP35 potently blocks innate immunity by inhibiting the host antiviral type-I interferon (IFN-I) system. However, regulation of VP35 function by cellular post-translational modifications is not understood. Our preliminary data suggest that EBOV replication is regulated by a novel mechanism involving VP35 ubiquitination by TRIM6, a host E3-ubiquitin ligase with known antiviral functions. Based on our data, we hypothesize that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination, while also blocking TRIM6 innate immune signaling activity. By using in vitro and in vivo approaches, including our newly generated TRIM6-knockout mice, we will test how TRIM6 contributes to EBOV replication and pathogenesis. We propose the following specific aims: 1) Determine the mechanistic role of VP35 ubiquitination in EBOV replication. We will use already available EBOV VP35 viral mutants that lack ubiquitination sites and test their function in relation to innate immune antagonism or viral polymerase function. 2) Determine the molecular mechanism by which TRIM6 regulates EBOV replication. We will examine how TRIM6 regulates EBOV replication by using TRIM6 knockout cell lines and in vitro ubiquitination and binding assays. We will also demonstrate that poly-Ub chains synthesized by TRIM6 in vivo promote EBOV replication, using our TRIM6-/- mice and mouse-adapted WT EBOV and the VP35 mutants that lack ubiquitination. The outcome of these studies is significant because it will provide fundamental knowledge about the mechanism of EBOV replication, which may apply to other viruses. Thus, a common approach for the development of broad-spectrum antivirals may be revealed. Importantly, this work will inform whether a host cellular factor, TRIM6, may be used as a target for the development of novel therapeutic strategy against one of the most lethal viruses of mankind.