Pediatric diarrheal diseases are a major cause of mortality among children under 5 years old. Rotavirus (RV) is the leading cause of diarrheal diseases in children, with an estimated 258 million cases and 200,000 deaths each year worldwide. Importantly, RV studies have long been used to establish a foundational understanding of enteric virus pathogenicity. Thus, in addition to representing a global health burden, RV provides a model from which we can gain insight into other viral processes. A hallmark of RV infection is dysregulation of Ca2+ signaling which underlies many aspects of enteric virus pathogenesis and disease. Until recently, this dysregulation was described as a monophasic increase in cytosolic Ca2+ levels. Using state-of-the-art live imaging, we found that RV infection results in distinct Ca2+ signal events that mimic previously characterized endogenous signals. These include intracellular Ca2+ puffs early in infection, and later “intercellular Ca2+ waves” that involve paracrine signaling from RV-infected to surrounding uninfected cells. RV nonstructural protein 4 (NSP4) is responsible for dysregulating Ca2+ homeostasis. Through distinct protein domains, RV NSP4 functions as a viroporin (VD), causing Ca2+ release from the endoplasmic reticulum, and an enterotoxin (ED), which elicits a receptor- dependent transient Ca2+ signal. While these domains are known to be involved in Ca2+ signal dysregulation, their relative contributions to replication and pathogenesis remain poorly defined. The overall objective of this research is to characterize NSP4 VD and ED functions, delineate how these domains contribute to the aberrant RV-induced Ca2+ signals and determine the role Ca2+ signaling plays in RV replication and pathogenesis. In Aim 1 we will characterize the role of NSP4 VD and ED in RV-induced Ca2+ signaling and RV replication. We have generated a panel of novel RV strains with targeted VD and ED mutations. Our extensive experience with RV reverse genetics, Ca2+ signaling, and enteric physiology make us well-suited to investigate this gap-in-knowledge. Further, our team first identified both NSP4 enterotoxin and NSP4 viroporin functions and is thus aptly poised to address these questions. We anticipate that most NSP4 VD mutants will attenuate early RV-induced Ca2+ signaling and viral replication. We generally expect mutations in NSP4 ED will attenuate later Ca2+ signaling, specifically paracrine signaling to adjacent cells. In Aim 2 we will determine the contributions of NSP4 VD and ED to RV pathogenicity ex vivo in human intestinal enteroids and in vivo in neonatal mice. We predict that through distinct functions, NSP4 VD and ED mutants will attenuate RV diarrhea and chloride secretion and will contribute to different aspects of pathogenicity. Collectively, these experiments establish new concepts about mechanisms of viral exploitation and mimicry of host signaling and pathophysiology, provide insight for improved vaccine efficacy, an...