The nonstructural glycoprotein of rotavirus affects intracellular calcium levels

Abstract

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirusinfected cells.

I, 2. Physiology and pathophysiology of the gut in relation to viral diarrhea

“…The nonstructural protein NSP4, expressed constitutively during rotavirus infection as well as added exogenously to noninfected cells, has been implicated in disturbances of Ca 2+ homeostasis. The expression of recombinant NSP4 protein in insect cells induced an increase in cytosol Ca 2+ concentration (Tian et al, 1994). This effect was proposed to be due to the release of Ca a+ from intracellular stores without increases in plasma membrane Ca 2÷ permeability and linked to a membrane destabilizing activity of NSP4 (Tian et al, 1995;Tian et al, 1996).…”

“…That a primary secretory component of rotavirus diarrhea might exist was first proposed based on the effect of rotavirus on Ca 2+ homeostasis of infected cells . The finding that the endogenous expression or the exogenous addition of the nonstructural protein NSP4 is capable of increasing [Ca2+] i (Dong et al, 1997;Tian et al, 1994;Tian et al, 1995) triggered a number of studies that lead to the hypothesis that this protein may act as a viral enterotoxin to induce secretion and diarrhea Estes and Morris, 1999;Morris and Estes, 2001). There is a series of evidence that supports this hypothesis: a) intraperitoneal or intraluminal injection of NSP4 or NSP4 peptide fragments cause age-dependent diarrhea in mice Sasaki et al, 2001); b) NSP4 and/or a fragment of this protein are released from infected cell monolayers as a consequence of secretion and apparently not cell lysis (Zhang et al, 2000); c) NSP4 or the synthetic NSP4~ 14-135 peptide potentiated cAMP-dependent C1-secretion in isolated mouse intestine mounted in Ussing chambers, similarly to carbachol ; d) exogenous addition of NSP4 or the NSP4114135 peptide to noninfected cells induced the release of Ca 2+ in HT29 cells through the activation of the phospholipase C-IP 3 cascade and a secondary increase in plasma membrane Ca 2+ permeability, similarly to cholinergic agents (Dong et al, 1997).…”

RNA silencing of rotavirus gene expression

“…A priori, one would expect that inhibition of the synthesis of NSP4, the ER membrane receptor for DLPs, should also lead to an accumulation of DLPs. However, since it is known that this protein alters the calcium homeostasis of cells (Tian et al, 1994(Tian et al, , 1995, its absence might have additional effects on the virus replication cycle. Silencing the NSP4 and VP7 genes should allow us to establish directly the role of these proteins in the translocation of DLPs into the lumen of the ER and in the removal of the intermediary lipid envelope.…”

Targeting of endoplasmic reticulum-associated proteins to axons and dendrites in rotavirus-infected neurons

“…In addition to its role as a receptor, NSP4 has been suggested to regulate the intracellular calcium levels of infected cells. This may promote the acquisition of correct protein configuration during virus maturation, but may also be involved in the cytotoxicity [45].…”