The Inositol Trisphosphate Receptor (IP R) is Dispensable for Rotavirus-induced Ca Signaling and Replication but Critical for Paracrine Ca Signals that Prime Uninfected Cells for Rapid Virus Spread.

Unlabelled: Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is an increase in cytosolic Ca caused by the nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that releases Ca from the endoplasmic reticulum (ER) and the increase in Ca signaling is critical for rotavirus replication. In addition to NSP4 itself, host inositol 1,4,5- trisphosphate receptor (IP R) ER Ca channels may contribute to rotavirus-induced Ca signaling and by extension, virus replication. Thus, we set out to determine the role of IP R Ca signaling during rotavirus infection using IP R-knockout MA104-GCaMP6s cells (MA104- GCaMP6s-IP R-KO), generated by CRISPR/Cas9 genome editing. Live Ca imaging showed that IP R-KO did not reduce Ca signaling in infected cells but eliminated rotavirus-induced intercellular Ca waves (ICWs) and therefore the increased Ca signaling in surrounding, uninfected cells. Further, MA104-GCaMP6s-IP R-TKO cells showed similar rotavirus susceptibility, single-cycle replication, and viral protein expression as parental MA104- GCaMP6s cells. However, MA104-GCaMP6s-IP R-TKO cells exhibited significantly smaller rotavirus plaques, decreased multi-round replication kinetics, and delayed virus spread, suggesting that rotavirus-induced ICW Ca 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 signals fully support rotavirus replication in individual cells; however, IP R is critical for rotavirus-induced ICWs and virus spread by priming Ca -dependent pathways in surrounding cells.

Importance: Many viruses exploit host Ca signaling to facilitate their replication; however, little is known about how distinct types of Ca signals contribute to the overall dysregulation of Ca signaling or promote virus replication. Using cells lacking IP R, a host ER Ca channel, we could differentiate between intracellular Ca signals within virus-infected cells and intercellular Ca waves (ICWs), which increase Ca signaling in neighboring, uninfected cells. In infected cells, IP R was dispensable for rotavirus-induced Ca signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca 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.