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 necess...