Oncosis in MA104 Cells Is Induced by Rotavirus Infection through an Increase in Intracellular Ca2+Concentration

Abstract

Rotavirus infection modifies the metabolism and ionic homeostasis of the host cell. First, there is an induction of viral synthesis with a parallel shutoff of cell protein production, followed by an increase of plasma membrane Ca2+ permeability, thereby inducing an increase of free cytoplasmic and sequestered Ca2+ concentrations. Cell death follows at a later stage. We studied the role of the increase in Ca2+ concentration in cell death. An elevation of extracellular Ca2+ concentration during infection induced an increase in [Ca2+]i and potentiated cell death. Buffering the increases in [Ca2+]i with BAPTA added at 6 h p.i. reduced the cytopathic effect without inhibiting viral protein synthesis and infectious particle production. Metoxyverapamil (D600), a Ca2+ channel inhibitor, added at 1 h p.i. reduced Ca2+ permeability, the increases in [Ca2+]i, and cell death produced by infection without modifying viral protein synthesis and infectious titer. Thapsigargin, the inhibitor of Ca(2+)-ATPase of endoplasmic reticulum, potentiated the increase of [Ca2+]i and accelerated the time course of cell death. Double staining with fluorescein diacetate and ethidium bromide or acridine orange and ethidium bromide showed that infected MA104 cells had lost plasma membrane integrity without DNA fragmentation or formation of apoptotic bodies. These results support the hypothesis that the increase in [Ca2+]i due to a product of viral protein synthesis triggers the chain of events that leads to cell death by oncosis.

Pathogenesis of Intestinal and Systemic Rotavirus Infection

“…This disruption of Ca 2ϩ homeostasis appears to be mediated by synthesis of viral proteins (17,49). Increased Ca 2ϩ permeability at both the plasma membrane and the endoplasmic reticulum leads to an increase in [Ca 2ϩ ] i , triggering a chain of events that leads to cell lysis (61 (19). In contrast, the increase in [Ca 2ϩ ] i induced by intracellular NSP4 is independent of PLC stimulation (73).…”

Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca 2+ Homeostasis Induced by Infection of Cultured Cells

“…However, for differentiated polarized cells, alternative modes of virion release without cell lysis have been proposed (11). Ca 2ϩ is known to control many key cell processes, and thus, its concentration within the cell is tightly regulated (6 homeostasis is concomitant with the onset of TPL assembly in the ER (24,28). Alterations in cell Ca 2ϩ homeostasis have been related to cytotoxicity and cell death, since the prevention or reduction of these changes results in a delay in cell death (28).…”

“…Ca 2ϩ is known to control many key cell processes, and thus, its concentration within the cell is tightly regulated (6 homeostasis is concomitant with the onset of TPL assembly in the ER (24,28). Alterations in cell Ca 2ϩ homeostasis have been related to cytotoxicity and cell death, since the prevention or reduction of these changes results in a delay in cell death (28). Furthermore, alterations in Ca 2ϩ homeostasis may be part of the basis of physiologic alterations in the intestine caused by rotavirus infection which finally lead to diarrhea (16,26).…”

Rotavirus Infection Induces an Increase in Intracellular Calcium Concentration in Human Intestinal Epithelial Cells: Role in Microvillar Actin Alteration

“…An increase of the intracellular sequestered Ca 2ϩ pools has also been measured (26,27). The effect of viral protein synthesis on [Ca 2ϩ ] i homeostasis may be responsible for cytopathic effect and cell death (26,29). Tian et al (35,36) have reported that the expression of the rotavirus nonstructural protein NSP4 increases [Ca 2ϩ ] i in Spodoptera frugiperda (Sf9) cells.…”