Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

J Virol. 2008 Nov;82(22):11331-43. doi: 10.1128/JVI.00577-08. Epub 2008 Sep 10.

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+).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Cell Line
  • Cell Membrane Permeability
  • Chlorocebus aethiops
  • Endoplasmic Reticulum / chemistry
  • Gene Expression*
  • Glycoproteins / genetics
  • Glycoproteins / metabolism*
  • Humans
  • Toxins, Biological / genetics
  • Toxins, Biological / metabolism*
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*

Substances

  • Glycoproteins
  • NS28 protein, rotavirus
  • Toxins, Biological
  • Viral Nonstructural Proteins
  • Calcium