Virus Research 167 (2012) 285–296 Contents lists available at SciVerse ScienceDirect Virus Research jo u r n al hom epa ge: www.elsevier.com/locate/virusres Dissecting the Ca 2+ entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells Yuleima Díaz 1 , Franshelle Pe˜ na, Olga Carolina Aristimu ˜ no, Lorena Matteo, Marisela De Agrela, Maria Elena Chemello, Fabian Michelangeli ∗∗ , Marie Christine Ruiz ∗ Laboratorio de Fisiología Gastrointestinal, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, Caracas 1020A, Venezuela a r t i c l e i n f o Article history: Received 15 March 2012 Received in revised form 13 May 2012 Accepted 16 May 2012 Available online 23 May 2012 Keywords: NSP4 Ca 2+ permeability ER stores Store-operated Ca 2+ entry (SOCE) Ca 2+ channel Na + /Ca 2+ exchanger (NCX) Rotavirus pathogenesis a b s t r a c t Rotavirus infection modifies Ca 2+ homeostasis provoking an increase in Ca 2+ permeation, cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyto ), total Ca 2+ pools and, a decrease of Ca 2+ response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca 2+ homeostasis is not yet known. It has been proposed that the increase in [Ca 2+ ] cyto is the result of Ca 2+ release from intracellular stores, thereby activating store-operated Ca 2+ entry (SOCE). We studied the mechanisms involved in the changes of Ca 2+ permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca 2+ stores induced by thapsigargin or ATP was not able to elicit Ca 2+ entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca 2+ ] cyto , is permeable to Mn 2+ and inhibited by La 3+ and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca 2+ entry is small and not modified by infection. The plasma membrane permeability to Ca 2+ in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB- R7943, an inhibitor of the plasma membrane Na + /Ca 2+ exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca 2+ entry. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Finely synchronized changes in cytosolic concentration of Ca 2+ modulate a variety of intracellular functions both in physiological conditions and pathological states. Viruses have evolved to utilize the universal Ca 2+ signals and to adapt or modify the cellular ionic environment to its advantage (Zhou et al., 2009). Rotavirus, as many other viruses interacts with Ca 2+ at different stages of its cycle, from the stability of its viral particle to the complex mechanisms of induction of diarrhea (Ruiz et al., 2000). Rotaviruses belonging to the Reoviridae family infect entero- cytes of the small intestine and represent the major etiological agent of viral gastroenteritis in children and young animals (Kapikian et al., 2001). The induction of diarrhea by rotavirus involves Ca 2+ -dependent secretory processes of mediators, water ∗ Corresponding author. Tel.: +58 212 5041164; fax: +58 212 5041093. ∗∗ Corresponding author. Tel.: +58 212 5041396; fax: +58 212 5041093. E-mail addresses: fabian@ivic.gob.ve, fmichelangeli@gmail.com (F. Michelangeli), mclr@ivic.gob.ve (M.C. Ruiz). 1 Present address: University of Bergen, Department of Molecular Biology, Høyteknologisenteret, Bioblokken, Thormøhlensgt. 55, 5008 Bergen, Norway. and electrolytes, as well as the induction of cell death in the differ- ent cell types that compose the intestinal epithelium (Michelangeli and Ruiz, 2003; Ruiz et al., 2000). The participation of the non- structural viral protein NSP4 functioning as a viral enterotoxin, and of the enteric nervous system in the pathogenesis of diarrhea rep- resent significant advances in its understanding (Ball et al., 1996; Lundgren et al., 2000; Morris and Estes, 2001). NSP4 and various recombinant and synthetic forms of NSP4 induce a phospholipase C-dependent elevation of the intracellular Ca 2+ concentration when added exogenously to cultured intestinal epithelial cells (Dong et al., 1997) and provoke diarrhea in animal models (Ball et al., 1996; Dong et al., 1997; Horie et al., 1999). The enterotoxigenic role, in vivo, of NSP4 is compatible with the observation of the secretion of NSP4 preferentially from the apical surface of infected Caco-2 cells (Bugarcic and Taylor, 2006) or of the truncated NSP4 peptide (NSP4 112–175 ) from rotavirus-infected MA-104 cells (Zhang et al., 2000). The soluble form of NSP4 or its putative peptide would diffuse within the intestinal lumen and interact with plasma mem- brane receptors on neighboring epithelial cells (Seo et al., 2008). Rotavirus infection of cultured cells induces change in the homeostasis of Ca 2+ mediated by the synthesis of viral pro- teins (Michelangeli et al., 1991; Perez et al., 1999; Ruiz et al., 2000). A progressive increase of Ca 2+ permeability of the plasma 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2012.05.012