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 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 microM) 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 x 10(-3) units/ml trypsin or 4.4 x 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.
Dong
Y
Y
Division of Molecular Virology, Baylor College of Medicine, Houston, TX 77030, USA.
Zeng
C Q
CQ
Ball
J M
JM
Estes
M K
MK
Morris
A P
AP
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000821:virology; D051379:Mice; D012401:Rotavirus; D012400:Rotavirus Infections / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D017361:Viral Nonstructural Proteins; C104282:nsp1 protein, Rotavirus; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D002118:Calcium