Ubiquitination during infection with Mouse Adenovirus

项目总结 病毒与宿主组件进行广泛的动态相互作用，通过以下方式促进感染 破坏细胞的内在和先天防御。病毒接管细胞过程的中心手臂依赖于 病毒利用细胞泛素系统来诱导宿主因子的降解。然而，有一个差距 在我们对泛素被病毒蛋白质利用的分子机制的理解中。在这里我们 建议对人和鼠腺病毒系统进行跨物种比较，以探索病毒如何 通过泛素颠覆宿主防御。人腺病毒5型(HAd5)是一种重要的核复制型DNA病毒 通过与两种病毒早期蛋白形成复合体来重定向细胞Cullin E3泛素连接酶的活性 (E1B55K和E4orf6)。我们最近开发了一种蛋白质组学方法来定义泛素化的宿主蛋白 表达了HAd5 E1B55K/E4orf6复合体。通过将我们的泛素组分析与整个细胞相结合 蛋白质组学，我们能够定义哪些底物泛素化，哪些随后被降解为 E1B55K/E4orf6复合体的结果。腺病毒感染的严格物种特异性限制了我们的能力 研究已经在其自然宿主，但鼠腺病毒1型(MAV-1)提供了另一种易处理的系统。 根据遗传相似性，MAV-1被认为编码HAd5的同源基因(mE1B55K和mE4orf6 复合体，这些蛋白质被认为以平行的方式重定向细胞泛素。我们已经申请了 我们的蛋白质组学流水线到MAV-1感染细胞，并使用全球泛素图谱来鉴定修饰的蛋白质 并被病毒降解。与HAd5不同，我们发现MAV-1独特地促进了 参与核酸传感和抗病毒干扰素信号传递的几种典型和非典型蛋白质， 包括PKR和STING。与HAd5 E1B55K/E4orf6复合体如何利用 用E1B55K组分选择泛素化底物时，我们惊讶地发现mE4orf6就足够了 为了以蛋白酶体和库林依赖的方式减少抗病毒RNA传感器PKR的丰度， 不依赖于mE1B55K。这些发现共同表明，在构成、机制上存在分歧。 HAd5和MAV-1导向的E3连接酶之间的组装和底物选择性。最重要的是 这意味着MAV-1和HAd5复合体以不同的方式利用泛素来中和内在和 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来 系统地定义内源性细胞泛素底物和相关的靶向通路 MAV-1感染。我们还将确定感染期间底物泛素化的功能后果。 在目标2中，我们将建立MAV-1导向的E3的组成和底物选择的机制 连接酶复合体并与HAd5比较。我们跨物种比较的结果将为我们提供对两者的洞察 管理腺病毒如何利用细胞泛素来分解宿主的核心原则和独特策略 防御和促进病毒致病。

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.