Joseph M. Hyser

Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is increased cytosolic Ca 2+ caused by nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that releases endoplasmic reticulum (ER) Ca 2+ , and the increased Ca 2+ signaling is critical for rotavirus replication. In addition to NSP4, host inositol 1,4,5-trisphosphate receptor (IP 3 R) ER Ca 2+ channels may contribute to rotavirus-induced Ca 2+ signaling and by extension, virus replication. Thus, we set out to determine the role of IP 3 R Ca 2+ signaling during rotavirus infection using CRISPR/Cas9 IP 3 R-knockout of MA104 cells stably expressing the GCaMP6s Ca 2+ indicator (MA104-GCaMP6s-IP 3 R-KO). Live Ca 2+ imaging showed that IP 3 R-KO did not reduce Ca 2+ signaling in infected cells but eliminated rotavirus-induced intercellular Ca 2+ waves (ICWs) and, therefore, the increased Ca 2+ signaling in surrounding, uninfected cells. MA104-GCaMP6s-IP 3 R-TKO cells showed similar rotavirus susceptibility, single-cycle replication, and viral protein expression as parental MA104-GCaMP6s cells. However, MA104-GCaMP6s-IP 3 R-TKO cells exhibited significantly smaller rotavirus plaques, decreased multi-round replication kinetics, and delayed virus spread, suggesting that rotavirus-induced ICW Ca 2+ signaling stimulates virus replication and spread. Inhibition of ICWs by blocking the purinergic receptor P2RY1 (P2Y1), which mediates the ICW Ca 2+ signals, also decreased rotavirus plaque size. Conversely, exogenous expression of P2Y1 in LLC-MK2-GCaMP6s cells, which natively lack P2Y1 and rotavirus ICWs, rescued the generation of rotavirus-induced ICWs and enabled plaque formation. In conclusion, this study shows that NSP4 Ca 2+ signals fully support rotavirus replication in individual cells; however, IP 3 R is critical for rotavirus-induced ICWs and virus spread by priming Ca 2+ -dependent pathways in surrounding cells. IMPORTANCE Many viruses exploit host Ca 2+ signaling to facilitate their replication; however, little is known about how Ca 2+ signals from different host and viral channels contribute to the overall dysregulation of Ca 2+ signaling or promote virus replication. Using cells lacking IP 3 R, a host ER Ca 2+ channel, we delineated intracellular Ca 2+ signals within virus-infected cells and intercellular Ca 2+ waves (ICWs), which increased Ca 2+</jats