Dysregulation of Endogenous and Paracrine Calcium Signaling Pathways by Rotaviruses and Caliciviruses

Abstract

Despite causing severe, potentially life‐threatening diarrhea, most enteric viruses only infect a small percent of intestinal epithelial cells. Thus, the induction of paracrine signaling pathways to dysregulate both infected and neighboring uninfected cell remains a major premise of enteric virus pathogenesis. However, virus‐induced paracrine signaling has never been directly observed and therefore the mechanisms have yet to be defined. Rotavirus (RV) remains a major cause of severe diarrhea in children worldwide. A hallmark of RV infection is the activation of aberrant calcium (Ca2+) signaling, which is necessary for replication and activation of secretory pathways in GI epithelium. We sought to characterize RV‐induced calcium signaling dynamics and identify paracrine signaling pathways responsible for infected‐to‐uninfected cell signaling. We conducted live‐cell Ca2+ imaging throughout the entire infection using cell lines and human intestinal enteroids (HIEs) engineered to stably express the genetically‐encoded Ca2+ indicator GCaMP. We found that rotavirus significantly increases both steady‐state and transient Ca2+ signaling mediated by RV nonstructural protein 4 (NSP4) and mutation of the NSP4 ion channel domain altered the RV‐induced Ca2+ signaling pattern observed, particularly low amplitude Ca2+ puffs observed early in infected cells. Further, isolated rotavirus‐infected cells generated multiple intercellular calcium waves (ICWs), which was the most prominent paracrine signal observed in both MA104 cells and HIEs. RV infection induces several signaling molecules, such as enterotoxin NSP4, prostaglandins and nitric oxide, and in many cell types extracellular purinergic signaling by ATP/ADP are responsible for ICW propagation. We found that RV‐induced ICWs were abolished by blocking extracellular ATP/ADP signaling with apyrase or purinergic receptor inhibitors or by CRISPR/Cas9‐mediated deletion of the P2Y1 receptor, but not by blocking extracellular enterotoxin form of NSP4, nitric oxide, or prostaglandin signaling. Rotavirus‐induced paracrine signaling was critical for multiple aspects of rotavirus pathogenesis, including fluid secretion and serotonin release, as well as regulating host responses, such as upregulation of IL‐1α and mucin secretion. Finally, we investigated whether activation of aberrant Ca2+ signaling and paracrine purinergic signaling were features of enteric caliciviruses using Tulane virus (TV), a rhesus monkey calicivirus. Like RV, we found that TV encodes for a viral ion channel in the endoplasmic reticulum and induces aberrant Ca2+ signaling during infection. TV infected cells also trigger ICWs that are blocked by purinergic signaling inhibitors. Thus, enteric viruses employ mechanisms to not only disrupt signaling in the virus‐infected cell but also exploit paracrine purinergic signaling to generate ICWs that represent a potent mechanism to amplify the pathophysiological signals underlying viral diarrhea.