Project Summary Enteroviruses are human pathogens that replicate in multiple organs and cause a variety of diseases, including gastroenteritis, pneumonia, myocarditis, and encephalitis. Currently, little is known about how enteroviruses alter the biology of infected cells. This proposed project, which is founded on our recent publication documenting the cellular proteins that enteroviral proteases target for cleavage, will elucidate the role of enteroviral proteases in changing the host cell enviornment during infection. The initial investigations of a subset of cleavage targets have so far yielded two candidate proteins, LSM14A and IRF2BP2, which activated innate immune signaling and inhibited viral replication in cultured cells. Our mechanistic studies have begun to reveal the molecular details of how these proteins limit virus infection and whether their innate immune functions are required for viral inhibition. These preliminary findings have built the foundation to characterize other host targets and determine their ability to regulate enterovirus infection. We have established a toolkit of novel methodologies and reagents that will enable the proposed work. For example, we have implemented an innovative experimental system that consists of human induced pluripotent stem cell-derived mesenchyme-free intestinal epithelial cells, which are highly susceptible to enteroviruses and respond to infection by triggering a robust innate immune program. Similarly, we have generated human cell lines containing interferon-stimulated genes tagged at their endogenous loci with fluorescent proteins, allowing real-time monitoring of immune induction in response to viral infections. We will employ these and other tools to investigate the molecular details of how the host target LSM14A activates innate immune signaling and inhibit viral infections (Aim 1); dissect the function of IRF2BP2 in the enterovirus lifecycle (Aim 2); and generate a comprehensive list of functionally important target proteins that have roles in enterovirus infection and/or antiviral innate immunity (Aim 3). Completion of these aims will reveal host restriction factors against enteroviral replication as well as their mechanisms of action. More broadly, this project will advance the understanding of enteroviral pathogenesis and potentially inform the design of broadly acting preventive and therapeutic strategies against enteroviral infections.