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腺病毒5（HAd 5）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 （E1 B55 K和E4 orf 6）。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HAd 5E 1B 55 K/E4 orf 6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 E1 B55 K/E4 orf 6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HAd 5的直系同源物（mE 1B 55 K和mE 4 orf 6 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HAd 5不同的是，我们发现MAV-1独特地促进HAd 5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HAd 5 E1 B55 K/E4 orf 6复合物如何利用 E1 B55 K组分来选择泛素化底物，我们惊奇地发现mE 4 orf 6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mE 1B 55 K无关。这些发现共同表明，在组成，机制， 组装和HAd 5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HAd 5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HAd 5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。