dsRNA production and sensing during DNA virus infection

DNA病毒感染过程中dsRNA的产生和传感

• 批准号: 10460518
• 负责人: Alexander Matthew Price
• 金额: $ 12.48万
• 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: 2021-08-03 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460518
• 关键词: 3' Untranslated Regions; Adenovirus Infections; Adenoviruses; Advisory Committees; Antibodies; Antiviral Response; Apoptosis; Base Pairing; Binding; Binding Proteins; Bioinformatics; Biology; Cell Nucleus; Cells; Cellular Stress; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Coupled; Cytoplasm; DNA; DNA Virus Infections; DNA Viruses; Data; Data Analyses; Double Stranded DNA Virus; Double-Stranded RNA; Environment; Exhibits; Faculty; Fluorescent in Situ Hybridization; Fractionation; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; Herpesviridae; Human Herpesvirus 4; Immune response; Immunoprecipitation; In Situ Hybridization; Infection; Innate Immune Response; Innate Immune System; Integration Host Factors; Interferons; K-Series Research Career Programs; Knowledge; Ligase; Location; Mass Spectrum Analysis; Mediating; Mentors; Messenger RNA; Modeling; Modernization; Modification; Molecular; Mutate; Nuclear; Outcome; Outcome Study; PRKR gene; Pathway interactions; Pattern; Pennsylvania; Phase; Play; Positioning Attribute; Process; Production; Property; Protein Biosynthesis; RNA; RNA Binding; RNA Processing; RNA Splicing; RNA Viruses; Regulation; Regulator Genes; Research; Research Personnel; Resolution; Ribonucleases; Role; Scientist; Seminal; Signal Transduction; Simplexvirus; Structure; System; Techniques; Technology; Testing; Training; Transcript; Universities; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Diseases; Work; adenosine deaminase; antagonist; base; career; ds RNA-Binding Proteins; ds-DNA; endonuclease; experience; experimental study; improved; inhibitor; insight; mutant; next generation sequencing; novel; oligoadenylate; pathogen; protein expression; receptor; response; sensor; skills; success; tool; transcriptome; transcriptome sequencing; virus host interaction

Project Summary Viral infections are known to produce double-stranded RNA (dsRNA), a molecule that is not present at high levels in uninfected host cells. This property of dsRNA is exploited by cells to sense viral infection and deploy anti-viral countermeasures. While DNA viruses produce viral mRNA molecules that look identical to cellular RNA, many DNA viruses are thought to produce dsRNA due to the process of symmetrical gene transcription of both strands of DNA. When we looked for the presence of dsRNA during adenovirus (AdV) infection using modern antibody-based techniques we found no evidence of dsRNA production, directly countering the existing dogma. Considering many DNA viruses encode antagonists of cellular dsRNA-sensing pathways, this directly calls into question the relevance of dsRNA sensing during DNA virus infection. While wildtype AdV did not produce detectable dsRNA, viral mutants which can no longer splice their own transcripts efficiently saw robust accumulation of dsRNA within the nucleus. Furthermore, these dsRNA-producing mutants activated cytoplasmic sensors of dsRNA such as PKR and RNaseL. The use of mutant viruses provides a unique opportunity to assess host responses to dsRNAs derived from DNA virus infection. Still, the question of how these nuclear dsRNAs are detected by cytoplasmic sensors remains unanswered. By completion of this mentored career development award I will gain training in RNA sequencing, quantitative mass spectrometry, and the bioinformatics approaches to analyze both. In the mentored phase I will continue my training with AdV, a relatively simple virus that provides powerful tools to understand regulation and sensing of DNA virus derived nuclear dsRNA. In the independent phase I will utilize herpes simplex virus (HSV- 1), a complex virus able to exert control over dsRNA-sensing pathways, as a model virus to study exploitation of dsRNA for viral gene regulation. This proposal will reveal the binding partners and localizations of DNA virus derived dsRNA as well as new strategies in which viruses exploit host cell gene regulatory machinery. In Aim 1 I will determine the localization and binding partners of viral dsRNA using immunoprecipitation coupled to next generation sequencing and mass spectrometry. These experiments will determine how nuclear dsRNA leads to activation of cytoplasmic sensors, as well as how AdV interacts with and blocks these novel pathways. In Aim 2 I will determine how HSV-1 regulates its own viral gene expression using the nuclear retention of overlapping viral transcript pairs that form dsRNA. The outcome of these experiments will reveal a new mechanism for viral gene regulation with broad implications for all herpesviruses. The outstanding training environment at CHOP and the University of Pennsylvania, coupled with the excellent advisory committee I have assembled, will greatly facilitate my research during the mentored phase as well as launch my career with the skills necessary to transition to an independent faculty position studying how host cells sense the RNAs generated by DNA viruses.

项目摘要 已知病毒感染会产生双链RNA（dsRNA），这是一种在细胞内不存在的分子。 在未感染的宿主细胞中含量更高。细胞利用dsRNA的这种特性来感知病毒感染， 部署抗病毒措施虽然DNA病毒产生的病毒mRNA分子看起来与 细胞RNA，许多DNA病毒被认为是由于对称基因的过程产生dsRNA， 两条DNA链的转录。当我们寻找腺病毒（AdV） 使用基于现代抗体的技术，我们没有发现直接产生dsRNA的证据 反对现有的教条。考虑到许多DNA病毒编码细胞dsRNA敏感的拮抗剂， 这直接质疑了DNA病毒感染期间dsRNA传感的相关性。而 野生型AdV不产生可检测的dsRNA，病毒突变体不再能够剪接它们自己的转录物 有效地看到dsRNA在细胞核内的稳健积累。此外，这些产生dsRNA的突变体 激活的dsRNA细胞质传感器，如PKR和RNaseL。突变病毒的使用提供了一种独特的 评估宿主对源自DNA病毒感染的dsRNA的应答的机会。但问题是 这些细胞核双链RNA被细胞质传感器检测到仍然没有答案。 通过完成这个指导职业发展奖，我将获得RNA测序方面的培训， 定量质谱法和生物信息学方法来分析两者。在指导阶段，我将 我继续接受AdV的培训，这是一种相对简单的病毒，它提供了强大的工具来理解监管， 检测DNA病毒衍生的核dsRNA。在独立的阶段，我将利用单纯疱疹病毒（HSV- 1），一种能够对dsRNA敏感途径施加控制的复杂病毒，作为模型病毒来研究利用 用于病毒基因调控的dsRNA。这一建议将揭示DNA病毒的结合伴侣和定位 衍生的dsRNA以及病毒利用宿主细胞基因调控机制的新策略。目标1 我将用免疫沉淀法确定病毒dsRNA的定位和结合伴侣， 代测序和质谱。这些实验将确定核dsRNA如何导致 细胞质传感器的激活，以及AdV如何与这些新途径相互作用并阻断这些新途径。在目标2 我将确定HSV-1如何利用重叠的细胞核保留来调节其自身的病毒基因表达。 形成双链RNA的病毒转录物对。这些实验的结果将揭示病毒感染的新机制。 基因调控对所有疱疹病毒具有广泛意义。CHOP出色的培训环境 和宾夕法尼亚大学，再加上我召集的优秀的咨询委员会，将大大 在指导阶段促进我的研究，并以必要的技能开始我的职业生涯， 过渡到一个独立的教师职位，研究宿主细胞如何感知DNA病毒产生的RNA。