Ubiquitination during infection with Mouse Adenovirus

项目摘要 病毒通过与宿主成分的广泛动态相互作用网络来促进感染， 破坏细胞内在和先天防御。病毒接管细胞过程的一个中心环节依赖于 病毒利用细胞泛素系统诱导宿主因子降解。然而， 我们对病毒蛋白利用泛素的分子机制的理解。这里我们 提出一个人类和小鼠腺病毒系统的跨物种比较，以探索病毒如何 通过泛素破坏宿主防御人腺病毒5（HAd5）是一种重要的核复制DNA病毒 通过与两种病毒早期蛋白形成复合物来重定向细胞Cullin E3泛素连接酶活性 （E1B55K和E4orf6）。我们最近开发了一种蛋白质组学方法来定义宿主蛋白质的泛素化， 表达HAd5E1B55K/E4orf6复合物。通过结合我们的泛素分析和全细胞 蛋白质组学，我们能够确定哪些底物是泛素化的，哪些底物随后被降解为 E1B55K/E4orf6复合物的结果。腺病毒感染的严格的物种特异性限制了我们的能力， 在其天然宿主中研究HAd，但小鼠腺病毒1型（MAV-1）提供了一种替代的易处理系统。 基于遗传相似性，MAV-1被认为编码HAd5的直系同源物（mE1B55K和mE4orf6 复合物，并且这些蛋白质被假定以平行方式重定向细胞泛素。我们应用 我们的蛋白质组学管道到MAV-1感染的细胞，并使用全局泛素分析来识别修饰的蛋白质， 并被病毒降解与HAd5不同的是，我们发现MAV-1独特地促进HAd5的降解。 涉及核酸传感和抗病毒干扰素信号传导的几种典型和非典型蛋白质， 包括PKR和STING。与HAd5 E1B55K/E4orf6复合物如何利用 E1B55K组分来选择泛素化底物，我们惊奇地发现mE4orf6足以 为了以蛋白酶体和Cullin依赖性方式减少抗病毒RNA传感器PKR的丰度， 与mE1B55K无关。这些发现共同表明，在组成，机制， 组装和HAd5与MAV-1定向的E3连接酶之间的底物选择性。一个总体 这意味着MAV-1和HAd5复合物以不同的方式利用泛素来抵消内源性和 先天免疫反应在目标1中，我们将利用多学科的定量蛋白质组学方法， 系统地定义内源性细胞泛素底物和相关的途径， MAV-1感染。我们还将确定感染过程中底物泛素化的功能后果。 在目的2中，我们将建立MAV-1定向E3的底物选择的组成和机制， 连接酶复合物并与HAd5比较。我们的跨物种比较结果将提供对这两个问题的见解。 核心原则和独特的策略，支配腺病毒如何利用细胞泛素拆除宿主 防御和促进病毒发病机制。

PROJECT SUMMARY Viruses exert an extensive network of dynamic interactions with host components to promote infection by dismantling cellular intrinsic and innate defenses. A central arm of viral takeover of cellular processes relies on viral exploitation of the cellular ubiquitin system to induce degradation of host factors. However, there is a gap in our understanding of the molecular mechanisms by which ubiquitin is harnessed by viral proteins. Here we propose a cross-species comparison of the human and mouse adenovirus systems to explore how viruses subvert host defenses via ubiquitin. Human adenovirus 5 (HAd5) is a prominent nuclear-replicating DNA virus that redirects cellular Cullin E3 ubiquitin ligase activity via complex formation with two viral early proteins (E1B55K and E4orf6). We recently developed a proteomics approach to define host proteins ubiquitinated when the HAd5 E1B55K/E4orf6 complex is expressed. By combining our ubiquitome analysis with whole cell proteomics, we were able to define which substrates are ubiquitinated and which are subsequently degraded as a result of the E1B55K/E4orf6 complex. The strict species-specificity of adenovirus infection limits our ability to study HAd in its natural host, but mouse adenovirus type 1 (MAV-1) provides an alternative tractable system. Based on genetic similarities, MAV-1 is thought to encode orthologs (mE1B55K and mE4orf6) to the HAd5 complex, and these proteins are presumed to redirect cellular ubiquitin in a parallel fashion. We have applied our proteomics pipeline to MAV-1 infected cells, and used global ubiquitin-profiling to identify proteins modified and degraded by the virus. Distinct from HAd5, we discovered that MAV-1 uniquely facilitates degradation of several canonical and non-canonical proteins involved in nucleic acid sensing and antiviral interferon signaling, including PKR and STING. Contrary to the prevailing dogma of how the HAd5 E1B55K/E4orf6 complex employs the E1B55K component to select ubiquitination substrates, we surprisingly discovered that mE4orf6 is sufficient to reduce abundance of the antiviral RNA sensor PKR in a proteasome- and Cullin- dependent manner, independent of mE1B55K. These findings collectively suggest divergence in the composition, mechanisms of assembly, and substrate selectivity between the HAd5 and MAV-1 directed E3 ligases. An overarching implication is that the MAV-1 and HAd5 complex exploit ubiquitin in different ways to counteract intrinsic and innate immune responses. In Aim 1 we will leverage a multidisciplinary, quantitative proteomics approach to systematically define the endogenous cellular ubiquitin substrates and associated pathways targeted during MAV-1 infection. We will also determine the functional consequences of substrate ubiquitination during infection. In Aim 2 we will establish the composition, and mechanisms of substrate selection for the MAV-1 directed E3 ligase complex and compare to HAd5. Results of our cross-species comparisons will provide insights into both core principles and distinct strategies that govern how adenoviruses exploit cellular ubiquitin to dismantle host defenses and facilitate viral pathogenesis.