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Journal of Virology, June 2008, p. 5815-5824, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02719-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca²⁺ Homeostasis Induced by Infection of Cultured Cells

José Luis Zambrano,¹ Yuleima Díaz,² Franshelle Peña,² Esmeralda Vizzi,¹ Marie-Christine Ruiz,² Fabián Michelangeli,² Ferdinando Liprandi,¹ and Juan Ernesto Ludert^1*

Center for Microbiology and Cell Biology,¹ Center for Biochemistry and Biophysics, Venezuelan Institute for Scientific Research (IVIC), Caracas 1020-A, Venezuela²

Received 21 December 2007/ Accepted 2 April 2008

Rotavirus infection of cells in culture induces major changes in Ca²⁺ homeostasis. These changes include increases in plasma membrane Ca²⁺ permeability, cytosolic Ca²⁺ concentration, and total cell Ca²⁺ content and a reduction in the amount of Ca²⁺ released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca²⁺ homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca²⁺ permeability of the plasma membrane and cytosolic Ca²⁺ concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca²⁺ permeability and Ca²⁺ pools released by agonist (ATP). In addition, the increase of total Ca²⁺ content (as measured by ⁴⁵Ca²⁺ uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca²⁺ homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca²⁺ homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects.

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* Corresponding author. Present address: Department of Experimental Pathology, Center for Research and Advanced Studies (CINVESTAV-IPN), Mexico City, 07360 Mexico. Phone: 52-57473800, ext. 5647. Fax: 52-55-57473377. E-mail: jeludert{at}gmail.com

Published ahead of print on 9 April 2008.

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Journal of Virology, June 2008, p. 5815-5824, Vol. 82, No. 12
0022-538X/08/$08.00+0     doi:10.1128/JVI.02719-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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