Enteric virus exploitation of calcium signaling

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Ca2+ signaling is a broadly conserved regulator of cell function, thus many viruses have evolved mechanisms to exploit Ca2+ signaling pathways to promote viral replication. Rotavirus (RV), which remains the leading cause of acute gastroenteritis and diarrheal deaths in children worldwide, is an archetypal example of this. RV infection induces global increases in cytosolic Ca2+ which both disrupts host cell physiology and facilitates RV replication. While global Ca2+ dysregulation has long been a hallmark of RV infection, the distinct types of signals that sustain this dysregulation, and thereby facilitate replication and pathogenesis, have not been fully identified. Recently, our lab resolved a novel RV-induced Ca2+ signal known as an intercellular Ca2+ wave (ICW), which is critically linked to both viral exploitation and host restitution. ICWs are mediated by paracrine ADP release from infected cells which binds to the P2Y1 receptor on neighboring cells, eliciting a cytosolic Ca2+ flux. Through this mechanism, RV dysregulates Ca2+ not only in infected cells, but also in neighboring, uninfected cells. We found that this P2Y1-mediated signal is critical to host responses that influence disease, including serotonin, chloride, and fluid secretion, and the expression of immune mediators. In preliminary studies, we have also found that ICWs enhance MAPK activation and stem cell markers, which contribute to restitution. However, inhibiting ICWs reduces RV shedding, suggesting that ICWs also facilitate RV replication. Importantly, our preliminary data also show that recombinant expression of RV non-structural protein 4 (NSP4), an endoplasmic reticulum localized viral Ca2+ channel, is sufficient to induce ICWs. Given this, our Central Hypothesis is that aberrant Ca2+ signaling caused by RV NSP4 initiates ADP release triggering paracrine activation of P2Y1 to regulate host epithelial responses to RV infection; however, RV exploits this P2Y1 signaling to prime surrounding cells for greater replication. To test this hypothesis, we will (Aim 1) characterize the RV-induced ADP release pathway from infected cells, (Aim 2) characterize the P2Y1-mediated proliferative and restitution responses to RV infection in the gut, and (Aim 3) determine how RV co-opts P2Y1 signaling to increase virus replication and spread. Given that a broad range of enteric pathogens disrupt Ca2+ signaling, completion of these aims will generate new mechanistic insights into the host/pathogen arms race, new understanding of pathogen recognition pathways, and identify new cellular targets for potentially broadly acting antiviral therapies.