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

Abstract

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 Ca²⁺-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 ([Ca²⁺]_i) was monitored in fura-2-loaded HT-29 cells using microscope-based fluorescence imaging. NSP4 (1 nM to 5 μM) induced both Ca²⁺ release from intracellular stores and plasmalemma Ca²⁺ influx. During NSP4-induced [Ca²⁺]_i mobilization, [Na⁺]_i homeostasis was not disrupted, demonstrating that NSP4 selectively regulated extracellular Ca²⁺ entry into these cells. The ED₅₀ of the NSP4 effect on peak [Ca²⁺]_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 [Ca²⁺]_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 (IP₃) production in an IP₃-specific radioreceptor assay. Taken together, these results suggest that NSP4 mobilizes [Ca²⁺]_i in human intestinal cells through receptor-mediated phospholipase C activation and IP₃ production.