dsRNA production and sensing during DNA virus infection

项目摘要 众所周知，病毒感染会产生双链RNA(DsRNA)，这种分子不存在于 未感染的宿主细胞中的高水平。DsRNA的这一特性被细胞利用来感知病毒感染和 部署反病毒对策。而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而产生dsrna。 双链DNA的转录。当我们在腺病毒(Adv)过程中寻找dsRNA的存在时 使用现代基于抗体的技术感染，我们没有发现直接产生dsRNA的证据 对抗现有的教条。考虑到许多DNA病毒编码细胞dsRNA传感的拮抗剂 这直接引起了人们对dsRNA检测在DNA病毒感染期间的相关性的质疑。而当 野生型adv不能产生可检测到的dsRNA，病毒突变体不再能剪接自己的转录本 有效地看到dsRNA在细胞核内强劲积累。此外，这些产生dsRNA的突变体 激活dsRNA的细胞质感受器，如PKR和RNASEL。突变病毒的使用提供了一种独特的 评估宿主对来自DNA病毒感染的dsRNA的反应的机会。尽管如此，问题是如何 这些核dsRNA是由细胞质传感器检测到的，但仍未得到回答。 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， 定量质谱学和生物信息学方法来分析两者。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，它提供了强大的工具来理解监管和 Dna病毒来源的核dsRNA的传感。在独立的第一阶段，将利用单纯疱疹病毒(HSV- 1)，一种能够控制dsRNA传感途径的复杂病毒，作为研究开发的模式病毒 DsRNA用于病毒基因调控。这一提议将揭示DNA病毒的结合伙伴和定位 衍生dsRNA以及病毒利用宿主细胞基因调控机制的新策略。在目标1中 我将使用与Next结合的免疫沉淀来确定病毒dsrna的定位和结合伙伴。 世代测序和质谱学。这些实验将确定核dsrna如何导致 细胞质感受器的激活，以及ADV如何与这些新途径相互作用并阻止它们。在AIM 2 我将确定HSV-1如何利用重叠的核保留来调节自己的病毒基因表达 形成dsRNA的病毒转录物对。这些实验的结果将揭示病毒的一种新机制 对所有疱疹病毒具有广泛影响的基因调控。CHOP卓越的培训环境 宾夕法尼亚大学，再加上我组建的优秀的咨询委员会，将极大地 在指导阶段促进我的研究，并用必要的技能启动我的职业生涯 过渡到研究宿主细胞如何感知DNA病毒产生的RNA的独立教员职位。

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.