Host IP
3
R channels are dispensable for rotavirus Ca
2+
signaling but critical for intercellular Ca
2+
waves that prime uninfected cells for rapid virus spread
Authors: Perry Jacob L. , Scribano Francesca J. , Gebert John T. , Engevik Kristen A. , Ellis Jenna M. , Hyser Joseph M. 
Published: December 2023

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+
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.