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. 1995 Sep;69(9):5763–5772. doi: 10.1128/jvi.69.9.5763-5772.1995

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

P Tian 1, M K Estes 1, Y Hu 1, J M Ball 1, C Q Zeng 1, W P Schilling 1
PMCID: PMC189437  PMID: 7637021

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)

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Selected References

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  1. Au K. S., Chan W. K., Burns J. W., Estes M. K. Receptor activity of rotavirus nonstructural glycoprotein NS28. J Virol. 1989 Nov;63(11):4553–4562. doi: 10.1128/jvi.63.11.4553-4562.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Au K. S., Mattion N. M., Estes M. K. A subviral particle binding domain on the rotavirus nonstructural glycoprotein NS28. Virology. 1993 Jun;194(2):665–673. doi: 10.1006/viro.1993.1306. [DOI] [PubMed] [Google Scholar]
  3. Bergmann C. C., Maass D., Poruchynsky M. S., Atkinson P. H., Bellamy A. R. Topology of the non-structural rotavirus receptor glycoprotein NS28 in the rough endoplasmic reticulum. EMBO J. 1989 Jun;8(6):1695–1703. doi: 10.1002/j.1460-2075.1989.tb03561.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  5. Bleasdale J. E., Bundy G. L., Bunting S., Fitzpatrick F. A., Huff R. M., Sun F. F., Pike J. E. Inhibition of phospholipase C dependent processes by U-73, 122. Adv Prostaglandin Thromboxane Leukot Res. 1989;19:590–593. [PubMed] [Google Scholar]
  6. Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem. 1987;56:395–433. doi: 10.1146/annurev.bi.56.070187.002143. [DOI] [PubMed] [Google Scholar]
  7. Carafoli E. The Ca2+ pump of the plasma membrane. J Biol Chem. 1992 Feb 5;267(4):2115–2118. [PubMed] [Google Scholar]
  8. Chan W. K., Au K. S., Estes M. K. Topography of the simian rotavirus nonstructural glycoprotein (NS28) in the endoplasmic reticulum membrane. Virology. 1988 Jun;164(2):435–442. doi: 10.1016/0042-6822(88)90557-0. [DOI] [PubMed] [Google Scholar]
  9. Cohen J., Laporte J., Charpilienne A., Scherrer R. Activation of rotavirus RNA polymerase by calcium chelation. Arch Virol. 1979;60(3-4):177–186. doi: 10.1007/BF01317489. [DOI] [PubMed] [Google Scholar]
  10. Crawford S. E., Labbé M., Cohen J., Burroughs M. H., Zhou Y. J., Estes M. K. Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells. J Virol. 1994 Sep;68(9):5945–5952. doi: 10.1128/jvi.68.9.5945-5952.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dormitzer P. R., Both G. W., Greenberg H. B. Presentation of neutralizing epitopes by engineered rotavirus VP7's expressed by recombinant vaccinia viruses. Virology. 1994 Oct;204(1):391–402. doi: 10.1006/viro.1994.1543. [DOI] [PubMed] [Google Scholar]
  12. Dormitzer P. R., Greenberg H. B. Calcium chelation induces a conformational change in recombinant herpes simplex virus-1-expressed rotavirus VP7. Virology. 1992 Aug;189(2):828–832. doi: 10.1016/0042-6822(92)90616-w. [DOI] [PubMed] [Google Scholar]
  13. Ericson B. L., Graham D. Y., Mason B. B., Hanssen H. H., Estes M. K. Two types of glycoprotein precursors are produced by the simian rotavirus SA11. Virology. 1983 Jun;127(2):320–332. doi: 10.1016/0042-6822(83)90147-2. [DOI] [PubMed] [Google Scholar]
  14. Estes M. K., Crawford S. E., Penaranda M. E., Petrie B. L., Burns J. W., Chan W. K., Ericson B., Smith G. E., Summers M. D. Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system. J Virol. 1987 May;61(5):1488–1494. doi: 10.1128/jvi.61.5.1488-1494.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Estes M. K., Graham D. Y., Smith E. M., Gerba C. P. Rotavirus stability and inactivation. J Gen Virol. 1979 May;43(2):403–409. doi: 10.1099/0022-1317-43-2-403. [DOI] [PubMed] [Google Scholar]
  16. Foder B., Scharff O., Thastrup O. Ca2+ transients and Mn2+ entry in human neutrophils induced by thapsigargin. Cell Calcium. 1989 Oct;10(7):477–490. doi: 10.1016/0143-4160(89)90025-0. [DOI] [PubMed] [Google Scholar]
  17. Graber R., Losa G. A. Subcellular localization and kinetic properties of phosphatidylinositol 4,5-bisphosphate phospholipase C and inositol phosphate enzymes from human peripheral blood mononuclear cells. Enzyme. 1989;41(1):17–26. doi: 10.1159/000469046. [DOI] [PubMed] [Google Scholar]
  18. Graber R., Losa G. A. Subcellular localization of inositide enzymes in established T-cell lines and activated lymphocytes. Anal Cell Pathol. 1993 Jan;5(1):1–16. [PubMed] [Google Scholar]
  19. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  20. Hu Y., Rajan L., Schilling W. P. Ca2+ signaling in Sf9 insect cells and the functional expression of a rat brain M5 muscarinic receptor. Am J Physiol. 1994 Jun;266(6 Pt 1):C1736–C1743. doi: 10.1152/ajpcell.1994.266.6.C1736. [DOI] [PubMed] [Google Scholar]
  21. Hu Y., Schilling W. P. Receptor-mediated activation of recombinant Trpl expressed in Sf9 insect cells. Biochem J. 1995 Jan 15;305(Pt 2):605–611. doi: 10.1042/bj3050605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hu Y., Vaca L., Zhu X., Birnbaumer L., Kunze D. L., Schilling W. P. Appearance of a novel Ca2+ influx pathway in Sf9 insect cells following expression of the transient receptor potential-like (trpl) protein of Drosophila. Biochem Biophys Res Commun. 1994 Jun 15;201(2):1050–1056. doi: 10.1006/bbrc.1994.1808. [DOI] [PubMed] [Google Scholar]
  23. Ludert J. E., Michelangeli F., Gil F., Liprandi F., Esparza J. Penetration and uncoating of rotaviruses in cultured cells. Intervirology. 1987;27(2):95–101. doi: 10.1159/000149726. [DOI] [PubMed] [Google Scholar]
  24. Margalit H., Spouge J. L., Cornette J. L., Cease K. B., Delisi C., Berzofsky J. A. Prediction of immunodominant helper T cell antigenic sites from the primary sequence. J Immunol. 1987 Apr 1;138(7):2213–2229. [PubMed] [Google Scholar]
  25. Meyer J. C., Bergmann C. C., Bellamy A. R. Interaction of rotavirus cores with the nonstructural glycoprotein NS28. Virology. 1989 Jul;171(1):98–107. doi: 10.1016/0042-6822(89)90515-1. [DOI] [PubMed] [Google Scholar]
  26. Michelangeli F., Ruiz M. C., del Castillo J. R., Ludert J. E., Liprandi F. Effect of rotavirus infection on intracellular calcium homeostasis in cultured cells. Virology. 1991 Apr;181(2):520–527. doi: 10.1016/0042-6822(91)90884-e. [DOI] [PubMed] [Google Scholar]
  27. Miller C. Ion channel structure and function. Science. 1992 Oct 9;258(5080):240–241. doi: 10.1126/science.1384128. [DOI] [PubMed] [Google Scholar]
  28. Petrie B. L., Estes M. K., Graham D. Y. Effects of tunicamycin on rotavirus morphogenesis and infectivity. J Virol. 1983 Apr;46(1):270–274. doi: 10.1128/jvi.46.1.270-274.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Poruchynsky M. S., Maass D. R., Atkinson P. H. Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER. J Cell Biol. 1991 Aug;114(4):651–656. doi: 10.1083/jcb.114.4.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Putney J. W., Jr Capacitative calcium entry revisited. Cell Calcium. 1990 Nov-Dec;11(10):611–624. doi: 10.1016/0143-4160(90)90016-n. [DOI] [PubMed] [Google Scholar]
  31. Putney J. W., Jr Inositol phosphates and calcium entry. Adv Second Messenger Phosphoprotein Res. 1992;26:143–160. [PubMed] [Google Scholar]
  32. Schilling W. P., Cabello O. A., Rajan L. Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca(2+)-influx pathway. Biochem J. 1992 Jun 1;284(Pt 2):521–530. doi: 10.1042/bj2840521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schilling W. P., Rajan L., Strobl-Jager E. Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics. J Biol Chem. 1989 Aug 5;264(22):12838–12848. [PubMed] [Google Scholar]
  34. Segrest J. P., De Loof H., Dohlman J. G., Brouillette C. G., Anantharamaiah G. M. Amphipathic helix motif: classes and properties. Proteins. 1990;8(2):103–117. doi: 10.1002/prot.340080202. [DOI] [PubMed] [Google Scholar]
  35. Segrest J. P., Jackson R. L., Morrisett J. D., Gotto A. M., Jr A molecular theory of lipid-protein interactions in the plasma lipoproteins. FEBS Lett. 1974 Jan 15;38(3):247–258. doi: 10.1016/0014-5793(74)80064-5. [DOI] [PubMed] [Google Scholar]
  36. Shahrabadi M. S., Babiuk L. A., Lee P. W. Further analysis of the role of calcium in rotavirus morphogenesis. Virology. 1987 May;158(1):103–111. doi: 10.1016/0042-6822(87)90242-x. [DOI] [PubMed] [Google Scholar]
  37. Shahrabadi M. S., Lee P. W. Bovine rotavirus maturation is a calcium-dependent process. Virology. 1986 Jul 30;152(2):298–307. doi: 10.1016/0042-6822(86)90133-9. [DOI] [PubMed] [Google Scholar]
  38. Shuttleworth T. J., Thompson J. L. Effect of temperature on receptor-activated changes in [Ca2+]i and their determination using fluorescent probes. J Biol Chem. 1991 Jan 25;266(3):1410–1414. [PubMed] [Google Scholar]
  39. Suzuki Y., Hruska K. A., Reid I., Alvarez U. M., Avioli L. V. Characterization of phospholipase C activity of the plasma membrane and cytosol of an osteoblast-like cell line. Am J Med Sci. 1989 Mar;297(3):135–144. doi: 10.1097/00000441-198903000-00001. [DOI] [PubMed] [Google Scholar]
  40. Tian P., Hu Y., Schilling W. P., Lindsay D. A., Eiden J., Estes M. K. The nonstructural glycoprotein of rotavirus affects intracellular calcium levels. J Virol. 1994 Jan;68(1):251–257. doi: 10.1128/jvi.68.1.251-257.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vickers J. D. U73122 affects the equilibria between the phosphoinositides as well as phospholipase C activity in unstimulated and thrombin-stimulated human and rabbit platelets. J Pharmacol Exp Ther. 1993 Sep;266(3):1156–1163. [PubMed] [Google Scholar]
  42. Yamada M., Yamada M., Richelson E. Role of signal transduction systems in neurotensin receptor down-regulation induced by agonist in murine neuroblastoma clone N1E-115 cells. J Pharmacol Exp Ther. 1993 Oct;267(1):128–133. [PubMed] [Google Scholar]
  43. Yule D. I., Tseng M. J., Williams J. A., Logdson C. D. A cloned CCK-A receptor transduces multiple signals in response to full and partial agonists. Am J Physiol. 1993 Nov;265(5 Pt 1):G999–1004. doi: 10.1152/ajpgi.1993.265.5.G999. [DOI] [PubMed] [Google Scholar]

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