期刊
JOURNAL OF VIROLOGY
卷 82, 期 22, 页码 11331-11343出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.00577-08
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资金
- Consejo Nacional de Investigaciones Cientificas y Tecnologicas (FONACIT), Venezuela [S1-2001000329, G2001000637, F2005000222]
- FONACIT-ECOSNORD [PI 2002000905]
- TOTAL
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 Ca-45(2+) uptake. Infection with SA11 clone 28 induced an increase in Ca2+ permeability and Ca-45(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 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 Ca-45(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 Ca2+ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca2+.
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