Project Summary/Abstract This proposal describes a four year career development plan and a research strategy for Dr. RobertOrchard to transition from a postdoctoral fellow to an independent academic faculty position investigating host-pathogen interactions. The mentored phase of the award (K99) will be completed under the continuedguidance of Dr. Herbert `Skip' Virgin in the Department of Pathology and Immunology at Washington UniversitySchool of Medicine. The overall research goal of the proposal is to determine molecular mechanisms ofnorovirus cofactors upon viral entry. Candidate: I have a long standing interest in understanding the molecular mechanisms underlyingcomplex host-pathogen interactions. I graduated summa cum laude from Texas A&M University with a degreein Microbiology. I subsequently joined the Molecular Microbiology Graduate Program at University of TexasSouthwestern Medical School. For my doctoral thesis in Dr. Neal Alto's laboratory, I described how bacterialvirulence proteins usurp the host cytoskeletal machinery and engineer pathogenic-signaling circuits within thecomplex environment of the cytoplasm of eukaryotic host cells. I then began a postdoctoral fellowship underthe mentorship of Dr. Skip Virgin. During my fellowship in Dr. Virgin's lab, it has been my goal to couple myexperiences with dissecting host-pathogen signaling networks with his ability to define the in vivo relevance ofhost-pathogen interactions in animal models. To this end, my research project has been focused onunderstanding the molecular mechanisms of murine norovirus (MNoV) replication and tropism due to its robustin vitro and in vivo systems. Specifically, we recently completed a whole-genome CRISPR screen for hostgenes required for MNoV replication. We discovered that MNoV binds a proteinaceous receptor, CD300lf, thatis necessary both in vitro and in vivo for MNoV replication and when expressed in human cells sufficient tobreak the species barrier of MNoV replication. Additionally, our work described a novel, unidentified cofactor inserum required for efficient MNoV binding to cells. This work is the foundation for the research proposaloutlined here. I plan to focus the remainder of my fellowship on obtaining professional skills and scientificinsight necessary to transition to a tenure-track position. Career Development Plan: During my final year as a postdoctoral fellow, I will focus a significantamount of effort (15%) to developing the professional skills challenging to me that are necessary for successfulindependent investigators. I have assembled a career advisory committee composed of Dr. Daved Fremont,Dr. Michael Diamond, and Dr. Thaddeus Stappenbeck that will evaluate my progress in overcomingdeficiencies in scientific writing and data presentation, mentoring, and laboratory management along with myscientific progress. Additionally, I will attend specific seminars both within and outside of WashingtonUniversity to enhance my training and preparation for transition to independence. Lastly, I have developed atimeline with specific milestones that will guide myself, my mentor Dr. Virgin, and my career advisorycommittee in preparing me for my goal of successfully competing for an independent RO1 grant at the end ofthis four year proposal. Research Project: Murine norovirus (MNoV) is an important model for understanding humannoroviruses (HNoVs) and for elucidating complex interactions between viruses, the host's microbiota, and theimmune system. Recent advances in HNoV culture systems have uncovered cofactors that promote viralreplication in vitro through currently unknown mechanisms. Here, we will continue our investigations into theinteractions between MNoV cofactors and receptors. More specifically, the experiments outlined will directlytest the novel hypothesis that norovirus tropism is determined by the combination of receptor and cofactorinteractions. Our preliminary data suggests an unexplored connection between cholesterol derived bile acids,the MNoV receptor CD300lf, and host derived ceramide lipid species in promoting viral entry. Importantly, it iswell established that the inability of both MNoV and HNoV to replicate in non-permissive cells are due todefects in viral entry. We will explore the interactions of each of these components in a combinatorial fashion.Furthermore, we will directly test the ability of MNoV to establish and maintain a persistent infection in micewhen these pathways are perturbed using genetic and pharmacological approaches. I anticipate that ourresults will reveal fundamental principles of norovirus entry and provide insights into establishing a robust andreproducible in vitro HNoV replication system. More broadly speaking our hypothesis, if proven true, has thepotential for establishing a novel approach for generating in vitro culture systems for currently uncultivableviruses. The initial findings of this project will help me transition to an independent academic position studyingthe interaction between noroviruses and their hosts. The completion of this project will not only provide novelinsights into norovirus biology but the framework for a competitive RO1 application.