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Journal of Virology logoLink to Journal of Virology
. 1996 Jul;70(7):4549–4557. doi: 10.1128/jvi.70.7.4549-4557.1996

Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions.

J Schalich 1, S L Allison 1, K Stiasny 1, C W Mandl 1, C Kunz 1, F X Heinz 1
PMCID: PMC190391  PMID: 8676481

Abstract

Recombinant subviral particles (RSPs) obtained by coexpression of the envelope (E) and premembrane (prM) proteins of tick-borne encephalitis virus in COS cells (S. L. Allison, K. Stadler, C. W. Mandl, C. Kunz, and F. X. Heinz, J. Virol. 69:5816-5820, 1995) were extensively characterized and shown to be ordered structures containing envelope glycoproteins with structural and functional properties very similar to those in the virion envelope. The particles were spherical, with a diameter of about 30 nm and a buoyant density of 1.14 g/cm3 in sucrose gradients. They contained mature E proteins with endoglycosidase H-resistant glycans as well as fully cleaved mature M proteins. Cleavage of prM, which requires an acidic pH in exocytic compartments, could be inhibited by treatment of transfected cells with ammonium chloride, implying a common maturation pathway for RSPs and virions. RSPs incorporated [14C]choline but not [3H]uridine, demonstrating that they contain lipid but probably lack nucleic acid. The envelope proteins of RSPs exhibited a native antigenic and oligomeric structure compared with virions, and incubation at an acidic pH (pH <6.5) induced identical conformational changes and structural rearrangements, including an irreversible quantitative conversion of dimers to trimers. The RSPs were also shown to be functionally active, inducing membrane fusion in a low-pH-dependent manner and demonstrating the same specific hemagglutination activity as whole virions. Tick-borne encephalitis virus RSPs thus represent an excellent model system for investigating the structural basis of viral envelope glycoprotein functions.

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Selected References

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  1. Allison S. L., Mandl C. W., Kunz C., Heinz F. X. Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR. Virus Genes. 1994 Jul;8(3):187–198. doi: 10.1007/BF01703077. [DOI] [PubMed] [Google Scholar]
  2. Allison S. L., Schalich J., Stiasny K., Mandl C. W., Kunz C., Heinz F. X. Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. J Virol. 1995 Feb;69(2):695–700. doi: 10.1128/jvi.69.2.695-700.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allison S. L., Stadler K., Mandl C. W., Kunz C., Heinz F. X. Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J Virol. 1995 Sep;69(9):5816–5820. doi: 10.1128/jvi.69.9.5816-5820.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. CLARKE D. H., CASALS J. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am J Trop Med Hyg. 1958 Sep;7(5):561–573. doi: 10.4269/ajtmh.1958.7.561. [DOI] [PubMed] [Google Scholar]
  5. Chambers T. J., Hahn C. S., Galler R., Rice C. M. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990;44:649–688. doi: 10.1146/annurev.mi.44.100190.003245. [DOI] [PubMed] [Google Scholar]
  6. Fonseca B. A., Pincus S., Shope R. E., Paoletti E., Mason P. W. Recombinant vaccinia viruses co-expressing dengue-1 glycoproteins prM and E induce neutralizing antibodies in mice. Vaccine. 1994;12(3):279–285. doi: 10.1016/0264-410x(94)90206-2. [DOI] [PubMed] [Google Scholar]
  7. Garoff H. Cross-linking of the spike glycoproteins in Semliki Forest virus with dimethylsuberimidate. Virology. 1974 Dec;62(2):385–392. doi: 10.1016/0042-6822(74)90400-0. [DOI] [PubMed] [Google Scholar]
  8. Guirakhoo F., Bolin R. A., Roehrig J. T. The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology. 1992 Dec;191(2):921–931. doi: 10.1016/0042-6822(92)90267-S. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guirakhoo F., Heinz F. X., Kunz C. Epitope model of tick-borne encephalitis virus envelope glycoprotein E: analysis of structural properties, role of carbohydrate side chain, and conformational changes occurring at acidic pH. Virology. 1989 Mar;169(1):90–99. doi: 10.1016/0042-6822(89)90044-5. [DOI] [PubMed] [Google Scholar]
  10. Guirakhoo F., Heinz F. X., Mandl C. W., Holzmann H., Kunz C. Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions. J Gen Virol. 1991 Jun;72(Pt 6):1323–1329. doi: 10.1099/0022-1317-72-6-1323. [DOI] [PubMed] [Google Scholar]
  11. Heinz F. X., Kunz C. Chemical crosslinking of tick-borne encephalitis virus and its subunits. J Gen Virol. 1980 Feb;46(2):301–309. doi: 10.1099/0022-1317-46-2-301. [DOI] [PubMed] [Google Scholar]
  12. Heinz F. X., Kunz C. Homogeneity of the structural glycoprotein from European isolates of tick-borne encephalitis virus: comparison with other flaviviruses. J Gen Virol. 1981 Dec;57(Pt 2):263–274. doi: 10.1099/0022-1317-57-2-263. [DOI] [PubMed] [Google Scholar]
  13. Heinz F. X., Kunz C. Isolation of dimeric glycoprotein subunits from tick-borne encephalitis virus. Intervirology. 1980;13(3):169–177. doi: 10.1159/000149122. [DOI] [PubMed] [Google Scholar]
  14. Heinz F. X., Kunz C. Protease treatment and chemical crosslinking of a flavivirus: tick borne encephalitis virus. Arch Virol. 1979;60(3-4):207–216. doi: 10.1007/BF01317492. [DOI] [PubMed] [Google Scholar]
  15. Heinz F. X., Stiasny K., Püschner-Auer G., Holzmann H., Allison S. L., Mandl C. W., Kunz C. Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology. 1994 Jan;198(1):109–117. doi: 10.1006/viro.1994.1013. [DOI] [PubMed] [Google Scholar]
  16. Heinz F. X., Tuma W., Guirakhoo F., Kunz C. A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of tick-borne encephalitis virus. J Biol Stand. 1986 Apr;14(2):133–141. doi: 10.1016/0092-1157(86)90032-6. [DOI] [PubMed] [Google Scholar]
  17. Holzmann H., Stiasny K., York H., Dorner F., Kunz C., Heinz F. X. Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions. Arch Virol. 1995;140(2):213–221. doi: 10.1007/BF01309857. [DOI] [PubMed] [Google Scholar]
  18. Konishi E., Mason P. W. Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein. J Virol. 1993 Mar;67(3):1672–1675. doi: 10.1128/jvi.67.3.1672-1675.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Konishi E., Pincus S., Fonseca B. A., Shope R. E., Paoletti E., Mason P. W. Comparison of protective immunity elicited by recombinant vaccinia viruses that synthesize E or NS1 of Japanese encephalitis virus. Virology. 1991 Nov;185(1):401–410. doi: 10.1016/0042-6822(91)90788-d. [DOI] [PubMed] [Google Scholar]
  20. Konishi E., Pincus S., Paoletti E., Laegreid W. W., Shope R. E., Mason P. W. A highly attenuated host range-restricted vaccinia virus strain, NYVAC, encoding the prM, E, and NS1 genes of Japanese encephalitis virus prevents JEV viremia in swine. Virology. 1992 Sep;190(1):454–458. doi: 10.1016/0042-6822(92)91233-k. [DOI] [PubMed] [Google Scholar]
  21. Konishi E., Pincus S., Paoletti E., Shope R. E., Burrage T., Mason P. W. Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection. Virology. 1992 Jun;188(2):714–720. doi: 10.1016/0042-6822(92)90526-u. [DOI] [PubMed] [Google Scholar]
  22. Konishi E., Pincus S., Paoletti E., Shope R. E., Wason P. W. Avipox virus-vectored Japanese encephalitis virus vaccines: use as vaccine candidates in combination with purified subunit immunogens. Vaccine. 1994 May;12(7):633–638. doi: 10.1016/0264-410x(94)90269-0. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  24. Lobigs M. Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6218–6222. doi: 10.1073/pnas.90.13.6218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mandl C. W., Guirakhoo F., Holzmann H., Heinz F. X., Kunz C. Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model. J Virol. 1989 Feb;63(2):564–571. doi: 10.1128/jvi.63.2.564-571.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mandl C. W., Heinz F. X., Kunz C. Sequence of the structural proteins of tick-borne encephalitis virus (western subtype) and comparative analysis with other flaviviruses. Virology. 1988 Sep;166(1):197–205. doi: 10.1016/0042-6822(88)90161-4. [DOI] [PubMed] [Google Scholar]
  27. Mason P. W., Pincus S., Fournier M. J., Mason T. L., Shope R. E., Paoletti E. Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection. Virology. 1991 Jan;180(1):294–305. doi: 10.1016/0042-6822(91)90034-9. [DOI] [PubMed] [Google Scholar]
  28. Pincus S., Mason P. W., Konishi E., Fonseca B. A., Shope R. E., Rice C. M., Paoletti E. Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis. Virology. 1992 Mar;187(1):290–297. doi: 10.1016/0042-6822(92)90317-i. [DOI] [PubMed] [Google Scholar]
  29. Pugachev K. V., Mason P. W., Frey T. K. Sindbis vectors suppress secretion of subviral particles of Japanese encephalitis virus from mammalian cells infected with SIN-JEV recombinants. Virology. 1995 May 10;209(1):155–166. doi: 10.1006/viro.1995.1239. [DOI] [PubMed] [Google Scholar]
  30. Randolph V. B., Winkler G., Stollar V. Acidotropic amines inhibit proteolytic processing of flavivirus prM protein. Virology. 1990 Feb;174(2):450–458. doi: 10.1016/0042-6822(90)90099-d. [DOI] [PubMed] [Google Scholar]
  31. Rey F. A., Heinz F. X., Mandl C., Kunz C., Harrison S. C. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature. 1995 May 25;375(6529):291–298. doi: 10.1038/375291a0. [DOI] [PubMed] [Google Scholar]
  32. Sato T., Takamura C., Yasuda A., Miyamoto M., Kamogawa K., Yasui K. High-level expression of the Japanese encephalitis virus E protein by recombinant vaccinia virus and enhancement of its extracellular release by the NS3 gene product. Virology. 1993 Feb;192(2):483–490. doi: 10.1006/viro.1993.1064. [DOI] [PubMed] [Google Scholar]
  33. Vorovitch M. F., Timofeev A. V., Atanadze S. N., Tugizov S. M., Kushch A. A., Elbert L. B. pH-dependent fusion of tick-borne encephalitis virus with artificial membranes. Arch Virol. 1991;118(1-2):133–138. doi: 10.1007/BF01311309. [DOI] [PubMed] [Google Scholar]
  34. Wengler G., Wengler G. Cell-associated West Nile flavivirus is covered with E+pre-M protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release. J Virol. 1989 Jun;63(6):2521–2526. doi: 10.1128/jvi.63.6.2521-2526.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]
  36. Winkler G., Heinz F. X., Kunz C. Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure. Virology. 1987 Aug;159(2):237–243. doi: 10.1016/0042-6822(87)90460-0. [DOI] [PubMed] [Google Scholar]
  37. Yamshchikov V. F., Compans R. W. Regulation of the late events in flavivirus protein processing and maturation. Virology. 1993 Jan;192(1):38–51. doi: 10.1006/viro.1993.1006. [DOI] [PubMed] [Google Scholar]

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