Craig B. Wilen, Ph.D. - US grants

Project Summary/Abstract This proposal describes a five-year career development program for the PI, Craig Wilen, M.D., Ph.D. to with the goal of preparing him for an independent research career as an academic physician-scientist. The PI graduated summa cum laude from Washington University in St. Louis with a degree in Biology and Economics. He then enrolled in the Medical Scientist Training Program at the University of Pennsylvania where he earned his M.D. and Ph.D. in Cell and Molecular Biology. Dr. Wilen continued his training as a resident physician in the Clinical Pathology Physician Scientist Training Program at Barnes-Jewish Hospital and Washington University. During his residency elective time, he embarked on basic science research in the lab of Dr. Herbert Virgin, who will serve as the research mentor in this proposal. Dr. Virgin is the Chair of the Department of Pathology and Immunology at Washington University and is a highly experienced and productive mentor of physician-scientists and a leading expert in viral pathogenesis and immunity. Dr. Wilen will continue his postdoctoral research in this lab. Washington University provides outstanding faculty members, collaborators, and core research facilities that will foster Dr. Wilen's scientific progress and career development. First, Dr. Wilen's Career Advisory Committee comprised of Drs. Diamond, Goldberg, Randolph, and Stappenbeck has extensive scientific expertise relevant to this proposal and highly successful track records as mentors. Second, the educational resources including the Office of Postdoctoral Affairs and the Division of Biology and Biomedical Sciences will enable Dr. Wilen to acquire and develop additional scientific and professional skills. Third, the research infrastructure within the Virgin lab and Washington University core facilities will enable Dr. Wilen to efficiently and skillfully address the scientific aims described herein. In summary, Washington University provides the ideal environment and resources for Dr. Wilen to develop and establish his independent career studying how viruses interact with the immune system to cause disease. The long-term goal of this study is to understand the role of virus-receptor interactions in governing murine norovirus tropism and pathogenesis in vivo. Murine norovirus is an important model for human norovirus, which is the primary cause of viral gastroenteritis worldwide. In addition, murine norovirus has been demonstrated to trigger inflammatory bowel disease in mice with certain genetic predispositions. However, the determinants of murine norovirus tropism and pathogenesis, and the mechanism of virus- induced enteric inflammation remain unknown. During Dr. Wilen's brief time in the Virgin lab, he performed a genome-wide CRISPR screen that identified CD300lf as a receptor for murine norovirus. Dr. Wilen is co-first author on a manuscript currently under review at Science describing this finding. This work has important implications for human norovirus infection and provides an unprecedented opportunity to study norovirus biology in vivo. In this proposal we will address the following aims: Aim1. We will test the hypothesis that CD300lf is necessary and sufficient for MNoV infection ex vivo and in vivo. Aim 2. We will test the hypothesis that CD300lf-dependent infection of macrophages, dendritic cells, and B cells differentially contributes to MNoV pathogenesis in vivo. These studies will define new mechanisms of murine norovirus pathogenesis, which has important implications for both human norovirus infection and enteric immunity.

Project Summary Human norovirus is the leading cause of acute gastroenteritis globally causing up to 200,000 deaths per year. However, there are no antiviral drugs or vaccines. We use the closely related mouse or murine norovirus (MNV) as a model system to understand how human norovirus causes infection and disease. More broadly, we also use MNV as a tool to uncover novel and fundamental aspects of how viruses interact with the host immune system and other intestinal microbes including bacteria and worms. We recently discovered CD300lf as a receptor of MNV. We leveraged this finding to identify tuft cells as a target cell of MNV in the mouse intestines. Tuft cells are rare epithelial cells that sense and initiate an immune response against intestinal worms. Tuft cells are also important in regulating intestinal inflammation, wound recovery, and several mucosal cancers. In preliminary data, we developed novel tuft cell deficient mice and both CD300lf conditional knockout and knock-in mice to study the role of tuft cells during acute and chronic norovirus infection. W We also developed an in vitro MNV tuft cell culture system that will enable us to dissect the complex interactions between norovirus, tuft cells, and the immune system. The objectives of this proposal are to determine the role of tuft cells in norovirus infection, to determine the immunological consequences of tuft cell infection, and to discover how infected tuft cells evade CD8+ T cell killing. In Aim 1, we will determine if viral infection impairs tuft cell function. We will identify the role of tuft cells in acute and persistent MNV infection. We will determine how MNV kills tuft cells during chronic infection, how infection perturbs the tuft cell transcriptome, and whether virus infection of tuft cells impairs the ability of tuft cells to protect the host against worm infection. In Aim 2, we will determine the mechanism by which tuft cells act as an immunoprivileged niche for MNV. MNV-specific CD8+ T cells ignore infected tuft cells during chronic infection yet they remain functional and able to detect antigen ex vivo. We will identify how tuft cell tropism enables resistance to CD8+ T cells by combining novel mouse lines, viral genetics, tuft cell and T cell co-culture, and adoptive transfer of both tuft cell and MNV-specific CD8+ T cells. In particular, we will determine if quiescent long-lived tuft cells, a tuft cell subtype, are a novel immunoprivileged reservoir for chronic infection. We anticipate this work will generate a detailed understanding about the cell tropism regulating norovirus transmission, the role of viral infection in type II immunity, and the mechanism by which norovirus evades immune CD8+ T cells. This is critical to our long-term goal of deciphering mechanisms of human norovirus pathogenesis and developing a successful human norovirus vaccine.