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Coronaviruses pp 175-186 | Cite as

The Role of Protease-Dependent Cell Membrane Fusion in Persistent and Lytic Infections of Murine Hepatitis Virus

  • Lee Mizzen
  • Maleki Daya
  • Robert Anderson
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 218)

Summary

We have defined three categories of cultured cell lines on the basis of their permissiveness (susceptibility to initial infection) to mouse hepatitis virus (MHV). Fully permissive L-2 cells gave rise to 100–1000-fold higher numbers of infectious centers than did semi-permissive LM, LM-K or C-1300 cells, whereas non-permissive Vero or C-6 cells were refractory to MHV infection. On an infected cell basis, there was no deficiency on the part of semi-permissive cell lines to replicate total viral RNA, viral polypeptides or progeny virions. Two of the semi-permissive cell lines (LM and LM-K) supported persistent MHV infection, while a third (C-1300) succumbed to lytic infection. LM and LM-K cells, but not C-1300 cells showed resistance to MHV-induced membrane fusion, even when placed in contact with fusion-active MHV-infected L-2 cells. The ability of a given cell to undergo fusion did not correlate with membrane lipid characteristics (unsaturated fatty acid and sterol content) which contribute to membrane “fluidity”. In order to more closely study the parameters of MHV-induced cell fusion, membranes were prepared from MHV-infected L-2 cells and monitored for their fusogenic potential with permissive L-2 cells, semi-permissive LM cells and non-permissive vero cells. Fusion was only observed with the permissive L-2 cells, and only when exogenous protease (trypsin or chymotrypsin) was added. When the membranes were prepared from 35S-methionine-labeled MHV-infected L-2 cells and subjected to protease treatment, the radiolabeled 180,000 dalton form of the E2-glycoprotein underwent proteolytic cleavage to yield a major product of approximately 90,000 daltons. Both trypsin and chymotrypsin were effective in this proteolytic cleavage and in activating membrane fusion. In a normally permissive, fusogenic infection of MHV in L-2 cells, the protease inhibitors TPCK and ZPCK, but not TLCK, were found to inhibit cell fusion. In MHV-infected L-2 cells, E2 was found almost exclusively as the 180,000 dalton form but turned over rapidly as shown by pulse-chase studies. TPCK and ZPCK but not TLCK inhibited turnover. The results suggest that L-2 cells contain a protease which cleaves at aromatic amino acids such as phenylalanine, and that this protease cleaves the 180,000 dalton form of the E2 to peptide fragments, one or more of which may activate cell fusion.

Keywords

Cell Fusion Sterol Content Mouse Hepatitis Virus Lytic Infection Murine Hepatitis Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Anderson, R., Cheley, S., and Haworth-Hatherell, E. (1979). Virology 97, 492–494.PubMedCrossRefGoogle Scholar
  2. Atkinson, P. (1973). in “Methods in Cell Biology”, ed. Prescott, D., Vol. 7, pp. 157–188, Academic Press, N.Y.Google Scholar
  3. Augusti-Tocco, G. and Sato, G. (1969). Proc. Natl. Acad. Sci. U.S.A. 64, 311–315.PubMedCrossRefGoogle Scholar
  4. Benda, P., Lightbody, J., Sato, G., Levine, L., and Sweet, S. (1968). Science 161, 370–371.PubMedCrossRefGoogle Scholar
  5. Buchmeier, M.J., Lewicki, H.A., Talbot, P.J., and Knobler, R.L. (1984). Virology 132, 261–270.PubMedCrossRefGoogle Scholar
  6. Cheever, F.S., Daniels, J.B., Pappenheimer, A.M., and Bailey, O.T. (1949) J. Exptl. Med. 90, 181–194.CrossRefGoogle Scholar
  7. Cheley, S., and Anderson, R. (1981). J. Gen. Virol. 54, 301–311.PubMedCrossRefGoogle Scholar
  8. Cheley, S. and Anderson, R. (1984) Anal. Biochem. 137, 15–19.PubMedCrossRefGoogle Scholar
  9. Cheley, S., Anderson, R., Cupples, M.J., Lee Chan, E.C.M., and Morris, V.L. (1981). Virology 112, 596–604.PubMedCrossRefGoogle Scholar
  10. Dubois-Dalcq, M.E., Doller, E.W., Haspel, M.V., and Holmes, K.V. (1982). Virology 119, 317–331.PubMedCrossRefGoogle Scholar
  11. Dulbecco, R., and Vogt, M. (1954). J. Exptl. Med. 99, 167–182.CrossRefGoogle Scholar
  12. Hirano, N., Goto, N., Ogawa, T., Ono, K., Murakami, T., and Fujiwara, K. (1980). Microbiol. Immunol. 24, 825–834.PubMedGoogle Scholar
  13. Haspel, M.V., Lampert, P.W., and Oldstone, M.B.A. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 4033–4036.PubMedCrossRefGoogle Scholar
  14. Kit, S., Dubbs, D.R., Piekarski, L.J., and Hsu, T.C. (1963). Exptl. Cell Res. 31, 297–312.PubMedCrossRefGoogle Scholar
  15. LePrevost, C, Virelizier, J.L., and Dupuy, J.M. (1975). J. Immunol. 115, 640–643.Google Scholar
  16. Lucas, A., Flintoff, W., Anderson, R., Percy, D., Coulter, M., and Dales, S. (1977). Cell 12, 553–560.PubMedCrossRefGoogle Scholar
  17. Manaker, R.A., Piczak, C.V., Miller, A.A., and Stanton, M.F. (1961). A J. Natl. Cancer Inst. 27, 29–51.Google Scholar
  18. Merchant, D.J., and Hellman, K.B. (1962). Proc. Soc. Exptl. Biol. Med. 110, 194–198.Google Scholar
  19. Mizzen, L., Cheley, S., Rao, M., Wolf, R., and Anderson, R. (1983). Virology 128, 407–417.PubMedCrossRefGoogle Scholar
  20. Nagashima, K., Wege, H., Meyermann, R., and ter Meulen, V. (1978). Acta Neuropathol. 45, 205–213.CrossRefGoogle Scholar
  21. Otsuki, K., and Tsubokura, M. (1981). Arch. Virol. 70, 315–320.PubMedCrossRefGoogle Scholar
  22. Rothfels, K.H., Axelrad, A.A., Siminovitch, L., McCulloch, E.A., and Parker, R.C. (1959). Can. Cancer Conf. 3, 189–214.Google Scholar
  23. Schoellmann, G. and Shaw, E. (1963). Biochem. 2, 252–255.CrossRefGoogle Scholar
  24. Segal, D.M., Powers, J.C., Cowen, G.H., Davies, D.R., and Wilcox, P.E. (1971). Biochem. 10, 3728–3738.CrossRefGoogle Scholar
  25. Shaw, E., Mares-Guia, M., and Cohen, W. (1965). Biochem. 10, 2219–2224.Google Scholar
  26. Sorensen, O., Coulter-Mackie, M.B., Puchalski, S., and Dales, S. (1984). Virology 137, 347–357.PubMedCrossRefGoogle Scholar
  27. Sorensen, O., Dugre, R., Percy, D., and Dales, S. (1982) Infect. Immun. 37, 1248–1260.PubMedGoogle Scholar
  28. Sorensen, O., Percy, D., and Dales, S. (1980). Arch. Neurol. 37, 478–484.PubMedCrossRefGoogle Scholar
  29. Stohlman, S.A., and Weiner, L.P. (1981). Neurol. 31, 38–44.CrossRefGoogle Scholar
  30. Storz, J., Rott, R., and Kaluza, G. (1981). Infect. Immun. 31, 1214–1222.PubMedGoogle Scholar
  31. Sturman, L.S., and Holmes, K.V. (1977). Virology 77, 650–660.PubMedCrossRefGoogle Scholar
  32. Sturman, L.S. and Holmes, K.V. (1984). In “Molecular Biology and Pathogenesis of Coronaviruses”, eds. Rottier, P.J.M, Van der Zeijst, B.A.M., Spaan, W.J.M. and Horzinek, M., pp. 25–35, Plenum Press, N.Y.Google Scholar
  33. Toth, T.E. (1982). Amer. J. Vet. Res. 43, 967–972.PubMedGoogle Scholar
  34. Wege, H., Siddell, S., and ter Meulen, V. (1982). Curr. Top. Microbiol. Immunol. 99, 165–200.PubMedCrossRefGoogle Scholar
  35. Weiner, L.P. (1973). Arch. Neurol. 28, 298–303.PubMedCrossRefGoogle Scholar
  36. Yasumura, Y., and Kawakita, Y. (1963). Nippon Rinsho (Japan) 21, 1209.Google Scholar
  37. Yoshikura, H., and Tejima, S. (1981). Virology 113, 503–511.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • Lee Mizzen
    • 1
  • Maleki Daya
    • 1
  • Robert Anderson
    • 1
  1. 1.Department of Microbiology and Infectious Diseases, Health Sciences CentreUniversity of CalgaryCalgaryCanada

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