Double-stranded RNA during DNA virus infection

DNA病毒感染期间的双链RNA

• 批准号: 10359055
• 负责人: Matthew D. Weitzman
• 金额: $ 60.48万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-05 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359055
• 关键词: Address; Adenovirus Infections; Adenoviruses; Antiviral Response; Apoptotic; Biogenesis; Biology; Cell Nucleus; Cells; Cessation of life; Code; Complex; DNA; DNA Virus Infections; DNA Viruses; Data; Double Effect; Double Stranded DNA Virus; Double-Stranded RNA; Enzymes; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Goals; Host Defense; Immune response; Infection; Innate Immune Response; Interferons; Knowledge; Lead; Ligase; Messenger RNA; Modeling; Modification; Molecular; Nuclear; Outcome; PRKR gene; Pathway interactions; Pattern; Production; Protein Biosynthesis; Proteins; Proteomics; RNA; RNA Binding; RNA Processing; RNA Viruses; RNA-Binding Proteins; Resources; Ribonucleases; Seminal; Signal Transduction; Source; Structure; System; Testing; Transcript; Translating; Translations; Ubiquitination; Viral; Viral Genome; Viral Physiology; Viral Proteins; Virus; Virus Diseases; Virus Replication; antagonist; antiviral drug development; ds-DNA; gene product; innate immune pathways; insight; mutant; oligoadenylate; pathogen; prevent; response; sensor; ubiquitin ligase; ubiquitin-protein ligase; viral DNA; viral RNA; virus host interaction

PROJECT SUMMARY Double-stranded (ds) RNA is an early danger signal that alerts the host to viral invasion and activates several innate immune pathways that limit virus replication. These pathways include type I and type III interferons that induce antiviral effectors, the oligoadenylate synthetase-ribonuclease L (OAS-RNase L) system that degrades ssRNA and leads to apoptotic death, and the protein kinase RNA dependent (PKR) pathway that halts protein synthesis. These host responses to dsRNA, and the many mechanisms viruses use to subvert the effector pathways, have been studied extensively in RNA viruses. However, there is a relative dearth of studies on dsRNA production during infection by DNA viruses, and there is a gap in our understanding of downstream effects or viral antagonism of antiviral pathways. Adenovirus (AdV) has a double-stranded DNA genome that has served as a powerful system for seminal discoveries in RNA biology, aided by availability of genetic mutants. AdV and other viruses with limited genome size and protein coding capacity have evolved to maximize gene expression through regulated transcription and use of both DNA strands for protein production. Thus, annealing of complementary single-stranded RNAs produced by symmetrical transcription of DNA virus genomes could lead to dsRNA. Although AdV is known to counter IFN responses and block PKR activation, it is not known whether infection generates dsRNA and how the antiviral dsRNA-activated pathways are evaded in infected cells. AdV mutants with early regions deleted have been useful for deciphering key viral functions required for infection. Deletion of early E1B and E4 genes results in unstable viral RNAs that are poorly transported and translated. The E1B55K and E4orf6 gene products form an E3 ubiquitin ligase required for efficient virus production. However, there is another gap in our understanding of how ubiquitination of substrates by this complex promotes RNA processing and late viral protein synthesis. We recently discovered that infection with AdV mutants that are defective for E1B55K or E4 generates dsRNA that accumulates in the nucleus, and that RNase L and PKR responses are activated. We also showed that a functional E1B55K/E4orf6 complex is required to prevent dsRNA during AdV infection and we have identified cellular RNA binding proteins that are ubiquitinated by the viral complex. These preliminary results have led to our overall hypothesis that the activity of the viral ligase ubiquitinates cellular RNA processing factors to prevent accumulation of dsRNA during infection and overcome antiviral host responses. Our Specific Aims are 1) to identify the source of dsRNA (viral or host), determine host responses activated, and 2) define how the E1B55K/E4orf6 ubiquitin ligase activity prevents antiviral responses to dsRNA. In this way we will use AdV as a model pathogen to study how dsRNA responses impact infection for DNA viruses. Our long-term goal is to uncover fundamental principles of gene expression by deciphering how DNA viruses manipulate RNA biogenesis pathways and evade antiviral defenses.

项目概要 双链 (ds) RNA 是一种早期危险信号，可提醒宿主病毒入侵并激活多种病毒 限制病毒复制的先天免疫途径。这些途径包括 I 型和 III 型干扰素 诱导抗病毒效应子，降解寡腺苷酸合成酶-核糖核酸酶 L (OAS-RNase L) 系统 ssRNA 会导致细胞凋亡，而蛋白激酶 RNA 依赖性 (PKR) 途径会停止蛋白质的合成 合成。这些宿主对 dsRNA 的反应，以及病毒用来破坏效应器的多种机制 途径，已在 RNA 病毒中进行了广泛研究。但相关研究相对缺乏 DNA病毒感染过程中dsRNA的产生，我们对下游的理解存在差距 抗病毒途径的作用或病毒拮抗作用。腺病毒 (AdV) 具有双链 DNA 基因组， 借助基因突变体的可用性，它已成为 RNA 生物学开创性发现的强大系统。 AdV 和其他基因组大小和蛋白质编码能力有限的病毒已经进化到最大化基因 通过调节转录和使用两条 DNA 链进行蛋白质生产来表达。因此，退火 DNA病毒基因组对称转录产生的互补单链RNA可以 导致 dsRNA。尽管已知 AdV 可以对抗 IFN 反应并阻断 PKR 激活，但尚不清楚 感染是否产生 dsRNA 以及感染者如何逃避抗病毒 dsRNA 激活途径 细胞。删除了早期区域的 AdV 突变体对于破译所需的关键病毒功能非常有用 感染。早期 E1B 和 E4 基因的缺失会导致病毒 RNA 不稳定，导致转运不良和 翻译的。 E1B55K和E4orf6基因产物形成高效病毒所需的E3泛素连接酶 生产。然而，我们对底物如何泛素化的理解还存在另一个差距。 复合物促进 RNA 加工和晚期病毒蛋白合成。我们最近发现，感染 E1B55K 或 E4 有缺陷的 AdV 突变体会​​产生在细胞核中积累的 dsRNA，并且 RNase L 和 PKR 反应被激活。我们还表明，功能性 E1B55K/E4orf6 复合物是 AdV 感染期间预防 dsRNA 所必需的，我们已经鉴定出细胞 RNA 结合蛋白 被病毒复合物泛素化。这些初步结果得出了我们的总体假设，即该活动 病毒连接酶泛素化细胞 RNA 加工因子，以防止 dsRNA 在 感染并克服宿主的抗病毒反应。我们的具体目标是 1) 确定 dsRNA 的来源（病毒 或宿主），确定激活的宿主反应，以及 2）定义 E1B55K/E4orf6 泛素连接酶活性如何 防止对 dsRNA 的抗病毒反应。这样我们将使用AdV作为模型病原体来研究dsRNA如何 反应会影响 DNA 病毒的感染。我们的长期目标是揭示基因的基本原理 通过破译 DNA 病毒如何操纵 RNA 生物发生途径并逃避抗病毒防御来表达。