The Caliciviridae family includes several human and animal enteric pathogens, including human noroviruses (HuNoV), which are a leading cause of viral diarrhea worldwide. Despite an enormous disease burden, little is known about the cellular or molecular signaling pathways responsible for HuNoV diarrhea. The disruption of host cell calcium (Ca2+) homeostasis by endoplasmic reticulum (ER)-targeted viral ion channels, called viroporins is a common feature of other enteric viruses, including rotavirus and enteroviruses. These proteins form Ca2+-conducting channels that releasing ER Ca2+ stores to activate host Ca2+ signaling pathways, particularly store-operated Ca2+ entry (SOCE) channels, like Orai1. SOCE Ca2+ signals are critical for virus replication and significantly contribute to pathogenesis. Despite many similarities between caliciviruses and other enteric viruses, whether caliciviruses also encode a viroporin is not known. We identified a putative viroporin domain in the NS1-2 protein, which may be analogous to that of the enterovirus viroporin 2B. The NS1-2 viroporin domain has the canonical cluster of lysine/arginine residues and an amphipathic alpha-helix, which are conserved between NoVs and Tulane virus (TV), closely related rhesus enteric caliciviruses (Recoviruses, ReCV) that we use as a model system for enteric caliciviruses. We predict that NS1-2 is a Ca2+-conducting viroporin analogous to enterovirus 2B. In preliminary studies, we found that TV NS1-2 protein has viroporin activity that maps to the putative viroporin domain. Using live Ca2+ imaging, we found that TV significantly elevates cytoplasmic Ca2+ signaling through a SOCE-mediated pathway and blockers of the Orai1 SOCE channel significantly attenuated the Ca2+ signaling and inhibited TV replication. Thus, we hypothesize that caliciviruses elevate cytosolic Ca2+ through viroporin-mediated release of ER Ca2+ and subsequent activation of SOCE, which promotes virus replication. We will address this hypothesis in two specific aims. Aim 1: Determine whether TV NS1-2 induces SOCE Ca2+ signaling as an ER-targeted viroporin. We will use live imaging to measure TV-induced Ca2+ signaling and STIM1 activation, and electrophysiology to characterize NS1-2 viroporin activity. Aim 2: Characterize the role of SOCE Ca2+ signaling in TV replication. We will define the mechanism by which Orai1 blockers inhibit TV replication using a range of classical and molecular virology assays. Our characterization of TV-induced SOCE Ca2+ signaling and the NS1-2 putative viroporin domain is conceptually innovative, and will establish a new paradigm in the calicivirus field by connecting it to Ca2+ signaling and viroporin-mediated pathophysiology. The proposed research is further significant because it will generate a foundation for future studies on HuNoV Ca2+ signaling using human intestinal enteroids and for translational studies on the therapeutic potential of Orai1 blockers as host-targeted antiviral therapies.