Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells.
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+).
Díaz
Yuleima
Y
Laboratorio de Fisiología Gastrointestinal, IVIC, Caracas 1020A, Venezuela.
Chemello
Maria Elena
ME
Peña
Franshelle
F
Aristimuño
Olga Carolina
OC
Zambrano
Jose Luis
JL
Rojas
Hector
H
Bartoli
Fulvia
F
Salazar
Leiria
L
Chwetzoff
Serge
S
Sapin
Catherine
C
Trugnan
Germain
G
Michelangeli
Fabian
F
Ruiz
Marie Christine
MC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000737:chemistry; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D014118:Toxins, Biological / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D002118:Calcium
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