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Journal of Virology, November 2008, p. 11331-11343, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.00577-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Expression of Nonstructural Rotavirus Protein NSP4 Mimics Ca²⁺ Homeostasis Changes Induced by Rotavirus Infection in Cultured Cells

Yuleima Díaz,¹ Maria Elena Chemello,¹ Franshelle Peña,¹ Olga Carolina Aristimuño,¹ Jose Luis Zambrano,² Hector Rojas,³ Fulvia Bartoli,⁴ Leiria Salazar,⁴ Serge Chwetzoff,⁵ Catherine Sapin,⁵ Germain Trugnan,⁵ Fabian Michelangeli,^1* and Marie Christine Ruiz^1*

Laboratorio de Fisiología Gastrointestinal,¹ Laboratorio de Biología de Virus,² Laboratorio de Fisiología Celular,³ Laboratorio de Biología Molecular, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, Caracas 1020A, Venezuela,⁴ INSERM U 538, CHU Saint Antoine, Université Pierre et Marie Curie, 75012 Paris, France⁵

Received 14 March 2008/ Accepted 28 July 2008

Rotavirus infection modifies Ca²⁺ homeostasis, provoking an increase in Ca²⁺ permeation, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), and total Ca²⁺ pools and a decrease in Ca²⁺ response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca²⁺ and the amount of Ca²⁺ sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca²⁺ pools were evaluated as ⁴⁵Ca²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca²⁺ permeability and ⁴⁵Ca²⁺ uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca²⁺ homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased ⁴⁵Ca²⁺ uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca²⁺.

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* Corresponding author. Mailing address for M. C. Ruiz: Laboratorio de Fisiología Gastrointestinal, IVIC, Caracas 1020A, Venezuela. Phone: 58 212 5041164. Fax: 58 212 5041093. E-mail: mclr{at}ivic.ve. Mailing address for F. Michelangeli: Laboratorio de Fisiología Gastrointestinal, IVIC, Caracas 1020A, Venezuela. Phone: 58 212 5041396. Fax: 58 212 5041093. E-mail: fabian{at}ivic.ve

Published ahead of print on 10 September 2008.

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Journal of Virology, November 2008, p. 11331-11343, Vol. 82, No. 22
0022-538X/08/$08.00+0     doi:10.1128/JVI.00577-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.