1887

Abstract

Flavivirus particles are synthesized in an immature form containing heterodimers of the proteins prM and E. Shortly before release from the cell, prM is cleaved by the host protease furin to yield mature virions. In this study, the furin-mediated cleavage of the tick-borne encephalitis (TBE) virus protein prM was prevented by specific mutagenesis of the cleavage site. This resulted in the production of immature TBE virions, which were shown to be completely non-infectious in BHK-21 cells. This finding contrasted with previous studies in which immature flavivirus particles produced by other techniques were shown to have considerable residual infectivity. The structural integrity of the mutant virus particles was confirmed by the characterization of physical and antigenic properties. Most importantly, infectivity could be restored by the addition of trypsin, which presumably cleaved protein prM at one of the monobasic sites retained in the mutated sequence. In the presence of trypsin, the mutant could be passaged repeatedly in BHK-21 cells, but if the protease was removed, the activated particles could initiate only a single round of infection, which again generated non-infectious virus progeny. These observations provide evidence that the infectivity of flaviviruses depends on the endoproteolytic cleavage of protein prM, which probably has a regulatory function rather than a direct role in virus entry. Moreover, the results illustrate that mutation of the furin cleavage site is a convenient way to produce single-round infectious flavivirus particles, which may be useful in vaccine and vector development.

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2003-01-01
2024-12-07
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References

  1. Allison S. L., Schalich J., Stiasny K., Mandl C. W., Kunz C., Heinz F. X. 1995; Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. J Virol 69:695–700
    [Google Scholar]
  2. Allison S. L., Schalich J., Stiasny K., Mandl C. W., Heinz F. X. 2001; Mutational evidence for an internal fusion peptide in flavivirus envelope protein E. J Virol 75:4268–4275
    [Google Scholar]
  3. Chambers T. J., Hahn C. S., Galler R., Rice C. M. 1990; Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649–688
    [Google Scholar]
  4. Clarke D. H., Casals J. 1958; Techniques for hemagglutination and hemagglutination inhibition with arthropod-borne viruses by antibody absorption. Am J Trop Med Hyg 7:561–573
    [Google Scholar]
  5. Davis N. L., Powell N., Greenwald G. F., Willis L. V., Johnson B. J., Smith J. F., Johnston R. E. 1991; Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cDNA clone. Virology 183:20–31
    [Google Scholar]
  6. Guirakhoo F., Heinz F. X., Mandl C. W., Holzmann H., Kunz C. 1991; Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions. J Gen Virol 72:1323–1329
    [Google Scholar]
  7. Guirakhoo F., Bolin R. A., Roehrig J. T. 1992; 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 191:921–931
    [Google Scholar]
  8. He R. T., Innis B. L., Nisalak A., Usawattanakul W., Wang S., Kalayanarooj S., Anderson R. 1995; Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2. J Med Virol 45:451–461
    [Google Scholar]
  9. Heidner H. W., McKnight K. L., Davis N. L., Johnston R. E. 1994; Lethality of PE2 incorporation into Sindbis virus can be suppressed by second-site mutations in E3 and E2. J Virol 68:2683–2692
    [Google Scholar]
  10. Heinz F. X., Kunz C. 1979; Protease treatment and chemical crosslinking of a flavivirus: tick borne encephalitis virus. Arch Virol 60:207–216
    [Google Scholar]
  11. Heinz F. X., Kunz C. 1981; Homogeneity of the structural glycoprotein from European isolates of tick-borne encephalitis virus: comparison with other flaviviruses. J Gen Virol 57:263–274
    [Google Scholar]
  12. Heinz F. X., Allison S. L. 2000; Structures and mechanisms in flavivirus fusion. Adv Virus Res 55:231–269
    [Google Scholar]
  13. Heinz F. X., Tuma W., Guirakhoo F., Kunz C. 1986; 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 14:133–141
    [Google Scholar]
  14. Heinz F. X., Mandl C. W., Holzmann H., Kunz C., Harris B. A., Rey F., Harrison S. C. 1991; The flavivirus envelope protein E: isolation of a soluble form from tick-borne encephalitis virus and its crystallization. J Virol 65:5579–5583
    [Google Scholar]
  15. Heinz F. X., Stiasny K., Puschner Auer G., Holzmann H., Allison S. L., Mandl C. W., Kunz C. 1994; Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology 198:109–117
    [Google Scholar]
  16. Hernandez L. D., Hoffman L. R., Wolfsberg T. G., White J. M. 1996; Virus–cell and cell–cell fusion. Annu Rev Cell Dev Biol 12:627–661
    [Google Scholar]
  17. Hsu M. C., Scheid A., Choppin P. W. 1987; Protease activation mutants of Sendai virus: sequence analysis of the mRNA of the fusion protein (F) gene and direct identification of the cleavage-activation site. Virology 156:84–90
    [Google Scholar]
  18. Iacono-Connors L. C., Smith J. F., Ksiazek T. G., Kelley C. L., Schmaljohn C. S. 1996; Characterization of Langat virus antigenic determinants defined by monoclonal antibodies to E, NS1 and preM and identification of a protective, non-neutralizing preM-specific monoclonal antibody. Virus Res 43:125–136
    [Google Scholar]
  19. Jain S. K., DeCandido S., Kielian M. 1991; Processing of the p62 envelope precursor protein of Semliki Forest virus. J Biol Chem 266:5756–5761
    [Google Scholar]
  20. Kielian M., Chatterjee P. K., Gibbons D. L., Lu Y. E. 2000; Specific roles for lipids in virus fusion and exit. Examples from the alphaviruses. Subcell Biochem 34:409–455
    [Google Scholar]
  21. Klenk H. D., Garten W. 1994; Activation cleavage of viral spike proteins by host proteases. In Cellular Receptors for Animal Viruses pp  241–279 Cold Spring Harbour, NY: Cold Spring Harbour Laboratory Press;
    [Google Scholar]
  22. Kofler R. M., Heinz F. X., Mandl C. W. 2002; Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence. J Virol 76:3534–3543
    [Google Scholar]
  23. Kopp A., Blewett E., Misra V., Mettenleiter T. C. 1994; Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus. J Virol 68:1667–1674
    [Google Scholar]
  24. Kuhn R. J., Zhang W., Rossmann M. G. 9 other authors & ; 2002; Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108:717–725
    [Google Scholar]
  25. Laemmli U. K., Favre M. 1973; Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80:575–599
    [Google Scholar]
  26. Lescar J., Roussel A., Wien M. W., Navaza J., Fuller S. D., Wengler G., Wengler G., Rey F. A. 2001; The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell 105:137–148
    [Google Scholar]
  27. Li Z., Sergel T., Razvi E., Morrison T. 1998; Effect of cleavage mutants on syncytium formation directed by the wild-type fusion protein of Newcastle disease virus. J Virol 72:3789–3795
    [Google Scholar]
  28. Lindenbach B. D., Rice C. M. 2001; Flaviviridae : The viruses and their replication. In Fields Virology , 4th edn. pp  991–1041 Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
    [Google Scholar]
  29. Lobigs M., Garoff H. 1990; Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62. J Virol 64:1233–1240
    [Google Scholar]
  30. McCune J. M., Rabin L. B., Feinberg M. B., Lieberman M., Kosek J. C., Reyes G. R., Weissman I. L. 1988; Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell 53:55–67
    [Google Scholar]
  31. Maisner A., Mrkic B., Herrler G., Moll M., Billeter M. A., Cattaneo R., Klenk H. D. 2000; Recombinant measles virus requiring an exogenous protease for activation of infectivity. J Gen Virol 81:441–449
    [Google Scholar]
  32. Mandl C. W., Heinz F. X., Kunz C. 1988; Sequence of the structural proteins of tick-borne encephalitis virus (Western subtype) and comparative analysis with other flaviviruses. Virology 166:197–205
    [Google Scholar]
  33. Mandl C. W., Heinz F. X., Stockl E., Kunz C. 1989; Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses. Virology 173:291–301
    [Google Scholar]
  34. Mandl C. W., Ecker M., Holzmann H., Kunz C., Heinz F. X. 1997; Infectious cDNA clones of tick-borne encephalitis virus European subtype prototypic strain Neudoerfl and high virulence strain Hypr. J Gen Virol 78:1049–1057
    [Google Scholar]
  35. Mandl C. W., Kroschewski H., Allison S. L., Kofler R., Holzmann H., Meixner T., Heinz F. X. 2001; Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo . J Virol 75:5627–5637
    [Google Scholar]
  36. Molloy S. S., Bresnahan P. A., Leppla S. H., Klimpel K. R., Thomas G. 1992; Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg–X–X–Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267:16396–16402
    [Google Scholar]
  37. Molloy S. S., Anderson E. D., Jean F., Thomas G. 1999; Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis. Trends Cell Biol 9:28–35
    [Google Scholar]
  38. Murray J. M., Aaskov J. G., Wright P. J. 1993; Processing of the dengue virus type 2 proteins prM and C-prM. J Gen Virol 74:175–182
    [Google Scholar]
  39. Nakayama K. 1997; Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J 27:625–635
    [Google Scholar]
  40. Plaimauer B., Mohr G., Wernhart W., Himmelspach M., Dorner F., Schlokat U. 2001; ‘Shed’ furin: mapping of the cleavage determinants and identification of its C-terminus. Biochem J 354:689–695
    [Google Scholar]
  41. Randolph V. B., Stollar V. 1990; Low pH-induced cell fusion in flavivirus-infected Aedes albopictus cell cultures. J Gen Virol 71:1845–1850
    [Google Scholar]
  42. Randolph V. B., Winkler G., Stollar V. 1990; Acidotropic amines inhibit proteolytic processing of flavivirus prM protein. Virology 174:450–458
    [Google Scholar]
  43. Rey F. A., Heinz F. X., Mandl C., Kunz C., Harrison S. C. 1995; The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution. Nature 375:291–298
    [Google Scholar]
  44. Salminen A., Wahlberg J. M., Lobigs M., Liljestrom P., Garoff H. 1992; Membrane fusion process of Semliki Forest virus. II. Cleavage-dependent reorganization of the spike protein complex controls virus entry. J Cell Biol 116:349–357
    [Google Scholar]
  45. Schalich J., Allison S. L., Stiasny K., Mandl C. W., Kunz C., Heinz F. X. 1996; Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions. J Virol 70:4549–4557
    [Google Scholar]
  46. Stadler K., Allison S. L., Schalich J., Heinz F. X. 1997; Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 71:8475–8481
    [Google Scholar]
  47. Stiasny K., Allison S. L., Schalich J., Heinz F. X. 2002; Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH. J Virol 76:3784–3790
    [Google Scholar]
  48. Wengler G., Wengler G. 1989; 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 63:2521–2526
    [Google Scholar]
  49. White J. M. 1992; Membrane fusion. Science 258:917–924
    [Google Scholar]
  50. Wool-Lewis R. J., Bates P. 1999; Endoproteolytic processing of the Ebola virus envelope glycoprotein: cleavage is not required for function. J Virol 73:1419–1426
    [Google Scholar]
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