Systematic dissection of viral genetic interactions and mutations with COEXPRES-Seq

Princeton University, Princeton NJ

Investigators

Abstract

Project Summary/Abstract In order to survive, a virus must manipulate the host cell environment, favoring its own replication while combating antiviral pathways. Viruses only encode a handful of genes, so most viral genes play multiple essential roles, and interact in order to form larger complexes with additional functions. Even subtle mutations can add a further layer of complexity, enabling different phenotypes to arise from closely related viral species. However, cataloging the functions of viral genes and their interactions remains unaccomplished for most viruses. Consequently, we cannot yet decipher the grammar by which viral genotype influences pathology and other complex phenotypes. I will perform comparative genomics to dissect the molecular basis of 4 strains of LCMV, which encode few genetic differences and yet cause distinct impacts on the liver and immune system. First, I will study the direct effect of infection on hepatocytes and dendritic cells (DCs) ex vivo. In addition to inoculating cells with each LCMV strain, I will glean mechanistic insights by pinpointing which gene(s) activate each cellular pathway. I propose to optimize COEXPRES-Seq (COmbinatorial EXpression Perturbation to Reveal Emergent Systems), a pooled genetic expression screen with a single-cell genomics readout. I will use this platform to quantify the contribution of each of the 4 LCMV genes, and their 15 combinations, in altering cell state (Aim 1). This study would be the first of its kind, as no study has systematically studied all combinations of a virus' genes. I expect to find emergent properties—pathways which are dysregulated only when, for example, both the WE Z and NP genes are expressed in hepatocytes. Next, I will study in vivo pathology. To explore the basis of WE-mediated hepatitis, I will challenge mice with WE or Arm strains, and perturb key pathways in the hepatocyte cell environment such as NF-κB and STAT1 (Aim 2). To better understand severe systemic disease, I will dually engraft mice with mouse or human liver and immune system and challenge these with both strains. By measuring hepatocyte and infiltrating immune cell states, I will identify differences between the two strains. Finally, I will investigate the molecular basis for DC infection and persistence of Arm/Cl13 and WE/Doc (Aim 3). I will develop molecular recorders of infection and inflammation, and associate patterns of these events with long-term changes in DC state. Together, this proposal is a first step in learning the rules that govern the viral genotype-phenotype relationship. My K99 training will be supervised by a distinguished mentoring committee: Drs. Alex Ploss (mentor), Britt Adamson (co-mentor), Yuri Pritykin (multiomics), and Juan C. de la Torre (LCMV), a vibrant intellectual environment. These individuals are experts in their fields, and will guide me as I learn new topics, such as state-of-the-art mouse models, genetic interactions, and trajectory analysis. In addition, I plan to further my growth through participation in conferences in systems biology and virology, courses of lab management, and writing workshops. The training in my K99 will launch me into an independent academic research position.