PROJECT SUMMARY / ABSTRACT This is a DP2 NIAID New Innovators Award proposal to harness cell-to-cell variability to understand viral infection outcomes at the single cell level. Infected cells differ in the outcome of infection (abortive/productive), the timing and level of viral gene expression and the number of progeny they produce. Two striking phenomena revealed by studying infection at the single cell level are (1) the ability of some cells to abort infection after viral gene expression has commenced and (2) the fact most infected cells release very few progeny while a small fraction of cells release thousands (here termed “super producers”). While it is now accepted that cell-to-cell variability affects infection outcome, we currently lack a molecular understating of the determinants of infection outcome in single cells, mainly due to the lack of appropriate tools and methodologies. Understanding these determinants is likely to lead to the discovery of novel cellular anti-viral modalities, better design of therapeutics and deepen our understanding of the viral life cycle. In this proposal I will apply cutting-edge technologies to investigate virus-host interactions at the single cell level and gain mechanistic insights as to the host factors that control the opposing phenotypes of abortive infection and super producers. My central hypothesis, supported by my past work and preliminary data using Herpes Simplex virus 1 (HSV-1) as a model system, is that infection outcome at the single cell level is dependent on the cell’s state at the time of infection and on the activation of specific cellular transcriptional programs. To test this hypothesis I will develop new approaches to interrogate virus-host interactions by combining viral genetics, single-cell RNA-sequencing, custom-made microfluidic devices, live- cell imaging and machine learning approaches. My background in performing cross-disciplinary research through the combination of classical virology, cell-biology, microfluidics and systems biology to study virus-host interactions uniquely positions me to successfully tackle these important questions. My results will identify the host factors that determine infection outcome and define a new paradigm, using cell-to-cell variability to understand virus-host interactions. The tools and technologies will be developed using HSV-1 as a model system, and then applied to investigate other important viral pathogens, in collaboration with leading virologists. This “high-risk high-reward” project requires a considerable investment of time and resources and the construction of new tools, making it a perfect fit for the DP2 program. The award will allow me to pursue cutting-edge science at the interface of virology and single cell biology and establish a long-term research plan to decipher the cellular mechanisms that control infection outcome.