Ubiquitination during infection with Mouse Adenovirus

小鼠腺病毒感染过程中的泛素化

• 批准号: 10364682
• 负责人: Matthew D. Weitzman
• 金额: $ 26.4万
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10364682
• 关键词: Adenovirus Infections; Adenoviruses; 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.

项目摘要 病毒与宿主成分建立了广泛的动态相互作用网络，以促进通过 拆除细胞内在和先天防御。病毒接管的中央部门依赖于 细胞泛素系统的病毒开发诱导宿主因子降解。但是，有差距 在我们对泛素通过病毒蛋白利用的分子机制的理解中。我们在这里 提出对人和小鼠腺病毒系统的跨物种比较，以探索病毒的方式 通过泛素颠覆宿主防御。人腺病毒5（HAD5）是一种突出的核重复DNA病毒 这将通过与两个病毒早期蛋白的复合物形成复合形成的细胞库蛋白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泛素化 表达了HAD5 E1B55K/E4ORF6复合物。通过将我们的泛元分析与全细胞结合在一起 蛋白质组学，我们能够定义哪些底物是泛素化的，哪些底物随后降解为 E1B55K/E4ORF6复合物的结果。腺病毒感染的严格物种特异性限制了我们的能力 研究具有天然宿主，但是小鼠腺病毒1型（MAV-1）提供了一种可替代的拖延系统。 基于遗传相似性，MAV-1被认为编码直系同源物（ME1B55K和ME4ORF6） 复合物，这些蛋白质被假定以平行方式重定向细胞泛素。我们已经申请了 我们的蛋白质组学管道到MAV-1感染的细胞，并使用全局泛素促进蛋白来鉴定经过修饰的蛋白质 并因病毒而降解。与HAD5不同，我们发现Mav-1独特地促进了降解 几种参与核酸感应和抗病毒干扰素信号传导的规范和非典型蛋白， 包括PKR和Sting。与HAD5 E1B55K/E4ORF6综合体如何使用的盛行的教条相反 E1B55K组件要选择泛素化底物，我们出人意料地发现ME4ORF6就足够了 为了减少抗病毒RNA传感器PKR的丰度，以蛋白酶体和cullin依赖性方式 独立于ME1B55K。这些发现统称在组成中的分歧，机制 组件和HAD5和MAV-1定向E3连接酶之间的底物选择性。总体 暗示是Mav-1和Mav-1复杂以不同的方式利用泛素来抵消固有和 先天免疫反应。在AIM 1中，我们将利用一种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针对性的相关途径 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AIM 2中，我们将建立MAV-1指示E3的底物选择的组成和机制 连接酶复合物，与HAD5进行比较。我们的跨物种比较的结果将为两者提供见解 核心原则和不同的策略，这些策略管理腺病毒如何利用细胞泛素来拆除宿主 防御和促进病毒发病机理。