The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production

Rotavirus infection is the leading cause of severe diarrhea in infants and young children worldwide. The rotavirus nonstructural protein NSP4 acts as a viral enterotoxin to induce diarrhea and causes Ca(2+)-dependent transepithelial Cl(−) secretion in young mice. The cellular basis of this phenomenon was investigated in an in vitro cell line model for the human intestine. Intracellular calcium concentration ([Ca(2+)](i)) was monitored in fura-2-loaded HT-29 cells using microscope-based fluorescence imaging. NSP4 (1 nM to 5 μM) induced both Ca(2+) release from intracellular stores and plasmalemma Ca(2+) influx. During NSP4-induced [Ca(2+)](i) mobilization, [Na(+)](i) homeostasis was not disrupted, demonstrating that NSP4 selectively regulated extracellular Ca(2+) entry into these cells. The ED(50) of the NSP4 effect on peak [Ca(2+)](i) mobilization was 4.6 ± 0.8 nM. Pretreatment of cells with either 2.3 × 10(−3) units/ml trypsin or 4.4 × 10(−2) units/ml chymotrypsin for 1–10 min abolished the NSP4-induced [Ca(2+)](i) mobilization. Superfusing cells with U-73122, an inhibitor of phospholipase C, ablated the NSP4 response. NSP4 induced a rapid onset and transient stimulation of inositol 1,4,5-trisphosphate (IP(3)) production in an IP(3)-specific radioreceptor assay. Taken together, these results suggest that NSP4 mobilizes [Ca(2+)](i) in human intestinal cells through receptor-mediated phospholipase C activation and IP(3) production.