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

Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is an increase in cytosolic Ca2+ caused by the nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that releases Ca2+ from the endoplasmic reticulum (ER) and the increase in Ca2+ signaling is critical for rotavirus replication. In addition to NSP4 itself, host inositol 1,4,5- trisphosphate receptor (IP3R) ER Ca2+ channels may contribute to rotavirus-induced Ca2+ signaling and by extension, virus replication. Thus, we set out to determine the role of IP3R Ca2+ signaling during rotavirus infection using IP3R-knockout MA104-GCaMP6s cells (MA104- GCaMP6s-IP3R-KO), generated by CRISPR/Cas9 genome editing. Live Ca2+ imaging showed that IP3R-KO did not reduce Ca2+ signaling in infected cells but eliminated rotavirus-induced intercellular Ca2+ waves (ICWs) and therefore the increased Ca2+ signaling in surrounding, uninfected cells. Further, MA104-GCaMP6s-IP3R-TKO cells showed similar rotavirus susceptibility, single-cycle replication, and viral protein expression as parental MA104- GCaMP6s cells. However, MA104-GCaMP6s-IP3R-TKO cells exhibited significantly smaller rotavirus plaques, decreased multi-round replication kinetics, and delayed virus spread, suggesting that rotavirus-induced ICW Ca2+ 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 Ca2+ signals fully support rotavirus replication in individual cells; however, IP3R is critical for rotavirus-induced ICWs and virus spread by priming Ca2+-dependent pathways in surrounding cells. ImportanceMany viruses exploit host Ca2+ signaling to facilitate their replication; however, little is known about how distinct types of Ca2+ signals contribute to the overall dysregulation of Ca2+ signaling or promote virus replication. Using cells lacking IP3R, a host ER Ca2+ channel, we could differentiate between intracellular Ca2+ signals within virus-infected cells and intercellular Ca2+ waves (ICWs), which increase Ca2+ signaling in neighboring, uninfected cells. In infected cells, IP3R was dispensable for rotavirus-induced Ca2+ signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP3R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca2+ 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.