Expression of Nonstructural Rotavirus Protein NSP4 Mimics Ca 2+ Homeostasis Changes Induced by Rotavirus Infection in Cultured Cells

Abstract

Rotavirus infection modifies Ca ²⁺ homeostasis, provoking an increase in Ca ²⁺ permeation, the cytoplasmic Ca ²⁺ concentration ([Ca ²⁺ ] _cyto ), and total Ca ²⁺ pools and a decrease in Ca ²⁺ 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 Ca ²⁺ and the amount of Ca ²⁺ 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 Ca ²⁺ pools were evaluated as ⁴⁵ Ca ²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca ²⁺ permeability and ⁴⁵ Ca ²⁺ 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 Ca ²⁺ 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 ⁴⁵ Ca ²⁺ 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 Ca ²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca ²⁺ .