rotavirus disrupts calcium homeostasis by nsp4 viroporin activity many viruses alter intracellular calcium homeostasis the rotavirus nonstructural protein 4 nsp4 an endoplasmic reticulum er transmembrane glycoprotein increases intracellular levels of cytoplasmic ca2 ca2cyto through a phospholipase cindependent pathway which is required for virus replication and morphogenesis however the nsp4 domain and mechanism that increases ca2cyto are unknown we identified an nsp4 domain amino acids aa 47 to 90 that inserts into membranes and has structural characteristics of viroporins a class of small hydrophobic viral proteins that disrupt membrane integrity and ion homeostasis to facilitate virus entry assembly or release mutational analysis showed that nsp4 viroporin activity was mediated by an amphipathic helical domain downstream of a conserved lysine cluster the lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity in epithelial cells expression of wildtype nsp4 increased the levels of free cytoplasmic ca2 by 37fold but nsp4 viroporin mutants maintained low levels of ca2cyto were retained in the er and failed to form cytoplasmic vesicular structures called puncta which surround viral replication and assembly sites in rotavirusinfected cells when ca2cyto was increased pharmacologically with thapsigargin viroporin mutants formed puncta showing that elevation of calcium levels and puncta formation are distinct functions of nsp4 and indicating that nsp4 directly or indirectly responds to elevated cytoplasmic calcium levels nsp4 viroporin activity establishes the mechanism for nsp4mediated elevation of ca2cyto a critical event that regulates rotavirus replication and virion assembly