Abstract
Mouse hepatitis virus (MHV), a murine coronavirus, utilizes murine carcinoembryonic antigens as receptors. The events that follow virus–receptor binding and eventually lead to virus entry are poorly understood. We studied the possible effects of MHV infection on intracellular calcium in a mouse astrocytoma cell line. Using the calcium-sensitive dye fluo-3 and confocal laser scanning microscopy, we found that MHV strain JHM induced an immediate (within 20 s) and transient (lasting no longer than 2 min) calcium increase in about 5% of the infected cells. The calcium increase was blocked by antibodies against the viral spike protein, suggesting that it was specifically triggered by the interaction of the viral spikes with cells. It was also inhibited by L-type calcium channel blockers and was not detected in calcium-free medium, suggesting that the calcium increase was caused by calcium influx from the extracellular medium. Studies of the kinetics of viral replication by immunofluorescence staining of the viral nucleocapsid protein revealed that at 3 h postinfection there was roughly the same percentage of cells (5%) that produced the viral protein as the percentage of cells that had responded with a calcium signal. This finding and the virus dilution studies together suggest that calcium responders may represent cells that had been infected with multiple viruses and undergone rapid viral replication. Furthermore, calcium channel blockers, including verapamil and cadmium chloride, and the calcium chelator EGTA inhibited virus infection. Therefore, the transient intracellular calcium increase reported here may be an early signaling event associated with virus infection.
Keywords: mouse hepatitis virus, mouse astrocytoma cells, intracellular calcium, fluo-3, confocal laser scanning microscopy, immunofluorescence
References
REFERENCES
- 1.Compton S.R., Barthold S.W., Smith A.L. Lab. Anim. Sci. 1993;43:15–28. [PubMed] [Google Scholar]
- 2.Williams R.K., Jiang G., Holmes K.V. Proc. Natl. Acad. Sci. USA. 1991;88:5533–5536. doi: 10.1073/pnas.88.13.5533. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Yokomori K., Lai M.M.C. J. Virol. 1992;66:6931–6938. doi: 10.1128/jvi.66.12.6931-6938.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Nedellec P., Dveksler G.S., Daniels E., Turbide C., Chow B., Basile A.A., Holmes K.V., Beauchemin N. J. Virol. 1994;68:4525–4537. doi: 10.1128/jvi.68.7.4525-4537.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Chen D.S., Asanaka M., Yokomori K., Wang F., Hwang S.B., Li H., Lai M.M.C. Proc. Natl. Acad. Sci. USA. 1995;92:12095–12099. doi: 10.1073/pnas.92.26.12095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Lai M.M.C., Cananagh D. Adv. Virus Res. 1997;48:1–100. doi: 10.1016/S0065-3527(08)60286-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Dreyer E.B., Kaiser P.K., Offermann J.T., Lipton S.A. Science. 1990;248:364–367. doi: 10.1126/science.2326646. [DOI] [PubMed] [Google Scholar]
- 8.Lo T.-M., Fallert C.J., Piser T.M., Thayer S.A. Brain Res. 1992;594:189–196. doi: 10.1016/0006-8993(92)91125-x. [DOI] [PubMed] [Google Scholar]
- 9.Hartshorn K.L., Collamer M., Auerbach M., Myers J.B., Pavlotsky N., Tauber A.I. J. Immunol. 1988;141:1295–1301. [PubMed] [Google Scholar]
- 10.Kornfeld H., Cruikshank W.W., Pyle S.W., Berman J.S., Center D.M. Nature. 1988;335:445–448. doi: 10.1038/335445a0. [DOI] [PubMed] [Google Scholar]
- 11.Dayanithi G., Yani N., Baghdiguian S., Fantini J. Cell Calcium. 1995;18:9–18. doi: 10.1016/0143-4160(95)90041-1. [DOI] [PubMed] [Google Scholar]
- 12.Michelangeli F., Liprandi F., Chemello M.E., Ciarlet M., Ruitz M.-C. J. Virol. 1995;69:3838–3847. doi: 10.1128/jvi.69.6.3838-3847.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Irurzun A., Arroyo J., Alvarez A., Carrasco L. J. Virol. 1995;69:5142–5146. doi: 10.1128/jvi.69.8.5142-5146.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Hirano N., Fujiwara K., Hino S., Matsumoto M. Arch. Gesamte Virusforsch. 1974;44:298–302. doi: 10.1007/BF01240618. [DOI] [PubMed] [Google Scholar]
- 15.Stohlman S.A., Brayton P.R., Fleming J.O., Weiner L.P., Lai M.M.C. J. Gen. Virol. 1982;63:265–275. doi: 10.1099/0022-1317-63-2-265. [DOI] [PubMed] [Google Scholar]
- 16.Fleming J.O., Stohlman S.A., Harmon R.C., Lai M.M.C., Frelinger J.A., Weiner L.P. Virology. 1983;131:296–307. doi: 10.1016/0042-6822(83)90498-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Burnier M., Centeno G., Burki E., Brunner H.R. Am. J. Physiol. 1994;266:C1118–C1127. doi: 10.1152/ajpcell.1994.266.4.C1118. [DOI] [PubMed] [Google Scholar]
- 18.Grynkiewicz G., Poenie M., Tsien R.Y. J. Biol. Chem. 1985;260:3440–3450. [PubMed] [Google Scholar]
- 19.Fleming J.O., Trousdale M.D., El-Zaatari F.A.K., Stohlman S.A., Weiner L.P. J. Virol. 1986;58:869–875. doi: 10.1128/jvi.58.3.869-875.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Huang R.C. J. Neurophysiol. 1993;70:1692–1703. doi: 10.1152/jn.1993.70.4.1692. [DOI] [PubMed] [Google Scholar]
- 21.Asanaka M., Lai M.M.C. Virology. 1993;197:732–741. doi: 10.1006/viro.1993.1649. [DOI] [PubMed] [Google Scholar]
- 22.Coutelier J.P., Godfraind C., Dveksler G.S., Wysocka M., Cardellichio C.B., Noel H., Holmes K.V. Eur. J. Immunol. 1994;24:1383–1390. doi: 10.1002/eji.1830240622. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Godfraind C., Langreth S.G., Cardellichio C.B., Knobler R., Coutelier J.P., Dubois-Dalcq M., Holmes K.V. Lab. Invest. 1995;73:615–627. [PubMed] [Google Scholar]
- 24.Hallett M.B., Fuchs P., Campbell A.K. Biochem. J. 1982;206:671–674. doi: 10.1042/bj2060671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Pace J., Hayman M.J., Galan J.E. Cell. 1993;72:505–514. doi: 10.1016/0092-8674(93)90070-7. [DOI] [PubMed] [Google Scholar]