Ubiquitination during infection with Mouse Adenovirus

小鼠腺病毒感染过程中的泛素化

• 批准号: 10152932
• 负责人: Matthew D. Weitzman
• 金额: $ 22万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-04 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152932
• 关键词: Adenovirus Infections; Adenoviruses; Antiviral Agents; Automobile Driving; Cell physiology; Cells; Complex; Cullin Proteins; DNA; DNA Damage; DNA Viruses; Data; Genetic; Goals; Host Defense; Human; Infection; Innate Immune Response; Integration Host Factors; Interferons; LATS1 gene; Lead; Ligase; Mediating; Modeling; Molecular; Mouse Protein; Mus; Nuclear; Nucleic Acids; Orthologous Gene; Pathway interactions; Process; Production; Proteins; Proteome; Proteomics; RNA; Reporting; Resources; Role; Seminal; Signal Transduction; Species Specificity; Substrate Specificity; System; Time; Ubiquitin; Ubiquitination; Viral; Viral Pathogenesis; Viral Proteins; Virus; Virus Diseases; YWHAQ gene; arm; base; cellular targeting; innate immune pathways; innate immune sensing; insight; member; multicatalytic endopeptidase complex; multidisciplinary; response; sensor; ubiquitin ligase; ubiquitin-protein ligase

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.

项目概要 病毒与宿主成分发挥广泛的动态相互作用网络，通过以下方式促进感染： 拆除细胞内在和先天的防御。病毒接管细胞过程的中心臂依赖于 病毒利用细胞泛素系统诱导宿主因子降解。然而，还有一个差距 帮助我们了解病毒蛋白利用泛素的分子机制。在这里我们 提出对人类和小鼠腺病毒系统进行跨物种比较，以探索病毒如何 通过泛素破坏宿主防御。人类腺病毒 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泛素化的宿主蛋白 表达 HAd5 E1B55K/E4orf6 复合物。通过将我们的泛素组分析与全细胞相结合 通过蛋白质组学，我们能够定义哪些底物被泛素化，哪些底物随后被降解为 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 研究自然宿主中的 HAd，但小鼠 1 型腺病毒 (MAV-1) 提供了另一种易于处理的系统。 基于遗传相似性，MAV-1 被认为编码 HAd5 的直向同源物（mE1B55K 和 mE4orf6） 复杂，并且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 我们针对 MAV-1 感染细胞的蛋白质组学流程，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，我们发现 MAV-1 独特地促进了 几种参与核酸传感和抗病毒干扰素信号传导的经典和非经典蛋白质， 包括 PKR 和 STING。与 HAd5 E1B55K/E4orf6 复合体如何使用的流行教条相反 E1B55K组件来选择泛素化底物，我们惊讶地发现mE4orf6就足够了 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， 独立于 mE1B55K。这些发现共同表明，其组成和机制存在差异。 HAd5 和 MAV-1 定向 E3 连接酶之间的组装和底物选择性。一个总体的 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在和 先天免疫反应。在目标 1 中，我们将利用多学科、定量蛋白质组学方法 系统地定义了内源性细胞泛素底物和相关的靶向途径 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 定向 E3 的组成和底物选择机制 连接酶复合物并与 HAd5 进行比较。我们的跨物种比较结果将提供对两者的见解 控制腺病毒如何利用细胞泛素来摧毁宿主的核心原则和独特策略 防御并促进病毒发病机制。