The Inositol Trisphosphate Receptor (IP3R) is Dispensable for Rotavirus-induced Ca2+Signaling and Replication but Critical for Paracrine Ca2+Signals that Prime Uninfected Cells for Rapid Virus Spread

Abstract Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is an increase in cytosolic Ca 2+ caused by the nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that releases Ca 2+ from the endoplasmic reticulum (ER) and the increase in Ca 2+ signaling is critical for rotavirus replication. In addition to NSP4 itself, 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 IP 3 R-knockout MA104-GCaMP6s cells (MA104- GCaMP6s-IP 3 R-KO), generated by CRISPR/Cas9 genome editing. 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. Further, 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 P2Y1 receptor also resulted in 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 distinct types of Ca 2+ signals 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 could differentiate between intracellular Ca 2+ signals within virus-infected cells and intercellular Ca 2+ waves (ICWs), which increase Ca 2+ signaling in neighboring, uninfected cells. In infected cells, IP 3 R was dispensable for rotavirus-induced Ca 2+ signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP 3 R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca 2+ signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This “pre-emptive priming” of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.