The rotavirus nonstructural glycoprotein NSP4 mobilizes Ca2+ from the endoplasmic reticulum

Abstract

We previously reported that expression of rotavirus nonstructural glycoprotein NSP4 is responsible for an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in Spodoptera frugiperda (Sf9) insect cells (P. Tian, Y. Hu, W. P. Schilling, D. A. Lindsay, J. Eiden, and M. K. Estes, J. Virol. 68:251-257, 1994). The purpose of the present study was to determine the mechanism by which NSP4 causes an increase in [Ca2+]i by measuring the permeability of the cytoplasmic and endoplasmic reticulum (ER) membranes in recombinant-baculovirus-infected Sf9 cells. No obvious change in plasmalemma permeability to divalent cations was observed in cells expressing NSP4 compared with that in cells expressing another rotaviral glycoprotein (VP7) when the influx of Ba2+, a Ca2+ surrogate, was monitored. The basal Ca2+ permeability of the internal Ca2+ store was evaluated by measuring the release of Ca2+ induced by ionomycin, a Ca2+ ionophore, or thapsigargin, an inhibitor of the ER Ca(2+)-ATPase pump, following suspension of the cells in Ca(2+)-free extracellular buffer. Releasable Ca2+ decreased with time to a greater extent in cells expressing NSP4 compared with that in cells expressing VP7, suggesting that NSP4 increases the basal Ca2+ permeability of the ER membrane. To determine the possible mechanism by which NSP4 increases ER permeability, purified NSP4 protein or a 22-amino-acid synthetic peptide consisting of residues 114 to 135 (NSP4(114-135) was added exogenously to noninfected Sf9 cells during measurement of [Ca2+]i. Both NSP4 and the NSP4(114-135 peptide produced a time-dependent increase in [Ca2+]i that was attenuated by prior inhibition of phospholipase C with U-73122. Pretreatment of the cells with thapsigargin completely blocked the increase in [Ca2+]i produced by NSP4(114-135, but the peptide only partially reduced the change in [Ca2+]i produced by thapsigargin. No changes in [Ca2+]i were seen in cells treated with control peptides. These results suggest that (i) exogenous NSP4 increases [Ca2+]i through the activation of phospholipase C, (ii) Ca2+ release by exogenous NSP4 is from a store that is a subset of the thapsigargin-sensitive compartment, and (iii) amino acid residues 114 to 135 of NSP4 are sufficient for this activity. In contrast to exogenous NSP4, the mechanism by which endogenously expressed NSP4 increases [Ca2+]1 appears to be unrelated to phospholipase C, since no effect of U-73122 was seen on the elevated [Ca2+]1 in cells expressing NSP4 and exogenously applied NSP4(114-135) caused a further increase in [Ca2+]1 in cells expressing NSP4 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Viral calciomics: Interplays between Ca2+ and virus

“…The rotavirus nonstructural glycoprotein NSP4 acts as a multifunctional enterotoxin [70] and has been shown to induce Ca 2+ mobilization in infected human epithelial cells through a PLC-dependent pathway [71]. Exogenously applied rotavirus nonstructural protein NSP4 can induce diarrhea in rodent pups and increases cytosolic Ca 2+ concentration via the activation of PLC and the resultant ER Ca 2+ depletion through IP 3 R [71]. In addition, endogenous NSP4 can also be secreted from the apical surface of polarized epithelial cells [72] or released outside after cell lysis, thus exerting exogenous action on neighboring non-infected cells.…”

“…Aside from the exogenous NSP4-induced, PLC-dependent Ca 2+ immobilization pathway (described above) [71], PLC-independent pathways also play important roles in inducing elevation of intracellular Ca 2+ [82]. The glycoprotein NSP4 is found to be primarily embedded in the ER membrane of rotavirus-infected cells [83].…”

“…The glycoprotein NSP4 is found to be primarily embedded in the ER membrane of rotavirus-infected cells [83]. Endogenously expressed NSP4 in Sf9 insect cells and HEK293 can alter the ER membrane permeability and therefore cause a sustained increase of cytosolic Ca 2+ concentration that is independent of the PLC pathway [71,82]. By using a liposome leakage assay that monitors the release of fluorescent dye from calnexin-loaded liposomes mimicking ER membrane, the membrane destabilization activity of NSP4 is mapped to residues 114-135 [84].…”

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.…”

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

“…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).…”