dsRNA production and sensing during DNA virus infection

DNA病毒感染过程中dsRNA的产生和传感

基本信息

批准号：
10893810
负责人：
Alexander Matthew Price
金额：
$ 24.9万
依托单位：
WISTAR INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-03 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10893810
关键词：
3&apos Untranslated Regions Adenovirus Infections Adenoviruses Advisory Committees Antibodies Antisense RNA 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 DNA biosynthesis Data 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 Learning Skill 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 Virus Replication Work antagonist career ds RNA-Binding Proteins ds-DNA dsRNA adenosine deaminase 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 viral detection virus host interaction

项目摘要

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），这是一种不存在于未感染的宿主细胞中的高水平。细胞利用DSRNA的这种特性，以感知病毒感染和部署抗病毒对策。而DNA病毒产生的病毒mRNA分子看起来与细胞RNA，由于对称基因的过程，许多DNA病毒被认为会产生DSRNA DNA的两个链的转录。当我们在腺病毒期间寻找dsRNA的存在时（ADV）使用现代抗体技术感染我们没有直接发现DSRNA的证据，直接反对现有的教条。考虑许多DNA病毒编码细胞dsRNA感应的拮抗剂途径，这直接引起了质疑DSRNA在DNA病毒感染过程中的相关性。尽管 WildType Adv没有产生可检测的DSRNA，病毒突变体，这些突变体无法再剪接自己的成绩单有效地看到了核中dsRNA的稳健积累。此外，这些产生DSRNA的突变体 DSRNA的活化细胞质传感器，例如PKR和RNASEL。使用突变病毒提供了独特的有机会评估对DNA病毒感染衍生的DSRNA的宿主反应。尽管如此，如何这些核DSRNA被细胞质传感器检测到未经检测。通过完成这个指导的职业发展奖，我将获得RNA测序的培训，定量质谱和生物信息学方法可以分析这两者。在指导阶段我将继续我的ADV培训，ADV是一种相对简单的病毒，提供了强大的工具来了解法规和传感DNA病毒衍生的核DSRNA。在独立阶段，I将利用单纯疱疹病毒（HSV- 1），一种能够控制DSRNA感应途径的复杂病毒，作为研究剥削的模型病毒 DSRNA用于病毒基因调节。该建议将揭示DNA病毒的结合伙伴和定位派生的DSRNA以及病毒利用宿主细胞基因调节机制的新策略。在目标1中我将使用免疫沉淀与接下来的免疫沉淀确定病毒DSRNA的定位和结合伴侣生成测序和质谱法。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如何与这些新型途径相互作用并阻止。在目标2中我将确定HSV-1如何使用重叠的核保留来调节其自身的病毒基因表达形成dsRNA的病毒转录对。这些实验的结果将揭示病毒的新机制基因调节对所有疱疹病毒具有广泛的影响。 CHOP的出色培训环境宾夕法尼亚大学，加上我组装的优秀咨询委员会，将大大在指导阶段促进我的研究，并以所需的技能来启动我的职业过渡到独立的教师职位，研究宿主细胞如何感知DNA病毒产生的RNA。