Live Cell Fluorescence Imaging Reveals Intercellular Calcium Waves and Chloride Channel Activation During Rotavirus Infection

Abstract

BackgroundRotavirus (RV) causes severe diarrheal disease in children worldwide. During infection, RV increases cytosolic calcium in intestinal epithelial cells through release of endoplasmic reticulum calcium and activation of the store‐operated calcium entry (SOCE) pathway. Increased cytosolic calcium is critical for RV replication and activation of secretory pathways in GI epithelium, yet the molecular details of these processes remain poorly defined. Thus, characterizing RV‐induced calcium signaling at the molecular level is needed to understand RV pathogenesis. We conducted live cell fluorescence imaging in cell lines and human intestinal enteroids (HIEs) as a biologically relevant model of the human small intestine to measure calcium signaling dynamics and chloride channel activation during RV infection.Methods & ResultsWe first engineered African green monkey kidney MA104 cells and HIEs to stably express GFP‐based genetically encoded calcium indicators to measure calcium dynamics throughout RV infection using live cell epifluorescence microscopy. We found that RV significantly increases the number and magnitude of calcium transients, which were attenuated with SOCE blockers. Furthermore, we observed that single RV‐infected cells triggered long‐distance intercellular calcium waves (ICWs) that encompassed surrounding uninfected cells in both MA104 cells and both duodenum and jejunum HIEs. Treatment with the ectoNTPase apyrase or purinergic receptor inhibitors blocked ICWs and reduced RV replication, mucin release, and serotonin secretion. These results indicate that extracellular ATP/ADP signaling elicits ICWs and is an important mediator of calcium dynamics during RV infection. Finally, we created HEK293 cell lines stably expressing halide‐sensitive YFP, a chloride channel biosensor, and either the calcium‐activated chloride channel anoctamin 1 (Ano1) or cystic fibrosis transmembrane conductance regulator (CFTR). We found that RV activated both mouse Ano1 and CFTR, which were attenuated with channel‐specific blockers, and suggests that multiple chloride channels can be activated during RV infection.ConclusionsOverall, our results support that RV causes dynamic calcium signaling during infection that is mediated by SOCE and purinergic signaling. Both pathways are potent stimulators of chloride secretion, thus they are potential therapeutic targets for developing life‐saving anti‐diarrheal drugs. Furthermore, we are the first to show that viruses can exploit purinergic signaling to generate ICWs that encompass uninfected cells and thereby potentially amplify the pathophysiological signaling important for diarrhea.Support or Funding InformationBCM Seed Funding, NIH F30 DK112563‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.