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Archives of Virology

, Volume 122, Issue 1–2, pp 77–94 | Cite as

Processing of dengue virus type 2 structural proteins containing deletions in hydrophobic domains

  • A. Gruenberg
  • P. J. Wright
Original Papers

Summary

The 5′ end of the genome of the dengue virus type 2 encoding the structural proteins was expressed using recombinant vaccinia virus. Three additional recombinants derived by deletion of selected dengue sequences within the parental construct were also expressed. They were designed to assess the role of hydrophobic domains in the processing of the viral polyprotein in intact cells. The first construct contained a deletion of nucleotides encoding most of the C protein; nucleotides encoding the hydrophobic domain at the carboxy terminus were retained. The second and third constructs contained smaller deletions of 72 bp and 129 bp encoding hydrophobic domains at the carboxy termini of C and prM respectively. Indirect immunofluorescence and radioimmunoprecipitation were used to detect prM and E in cells infected with recombinant viruses. The results showed that deletion of 90% of C had no apparent effect on the processing of prM and E, and that the signal sequence for E at the carboxy terminus of prM was active in the absence of the upstream signal sequence for prM at the carboxy terminus of C. Deletion of the hydrophobic sequences preceding the amino terminus of E prevented cleavage at the prM-E junction. These results obtained using infected cells were consistent with the published findings for the translation of flavivirus RNA in vitro, and indicated the importance of membrane association in the cleavage of structural proteins from the flavivirus polyprotein. In addition, cells infected with the recombinant virus containing the large deletion in the C coding region released the E glycoprotein into the culture medium.

Keywords

Signal Sequence Vaccinia Virus Indirect Immunofluorescence Dengue Virus Vaccinia 
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.

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References

  1. 1.
    Bell JR, Kinney RM, Trent DW, Lenches EM, Dalgarno L, Strauss JH (1985) Aminoterminal amino acid sequences of structural proteins of three flaviviruses. Virology 143: 224–229Google Scholar
  2. 2.
    Biedryzcka A, Cauchi MR, Bartholomeusz A, Gorman JJ, Wright PJ (1987) Characterization of protease cleavage sites involved in the formation of the envelope glycoprotein and three non-structural proteins of dengue virus type 2 New Guinea C strain. J Gen Virol 68: 1317–1326Google Scholar
  3. 3.
    Bonner WM, Laskey RA (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46: 83–88Google Scholar
  4. 4.
    Boulton RW, Westaway EG (1977) Togavirus RNA: reversible effect of urea on genomes and absence of subgenomic viral RNA in Kunjin virus-infected cells. Arch Virol 55: 201–208Google Scholar
  5. 5.
    Boyle DB, Couper BE, Both GW (1984) Multiple-cloning-site plasmids for the rapid construction of recombinant poxiviruses. Gene 35: 169–177Google Scholar
  6. 6.
    Bray M, Zhao B, Markoff L, Eckels KH, Chanock RM, Lai C-J (1989) Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS 1 are protected against fatal dengue virus encephalitis. J Virol 63: 2853–2856Google Scholar
  7. 7.
    Castle E, Nowak T, Leidner U, Wengler G, Wengler G (1985) Sequence analysis of the viral core protein and the membrane-associated proteins V 1 and NV 2 of the flavivirus West Nile virus and of the genome sequence for these proteins. Virology 145: 227–236Google Scholar
  8. 8.
    Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, Brandt WE (1989) Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J Gen Virol 70: 37–43Google Scholar
  9. 9.
    Coia G, Parker MD, Speight G, Byrne ME, Westaway EG (1988) Nucleotide and complete amino acid sequences of Kunjin virus: definitive gene order and characteristics of the virus-specified proteins. J Gen Virol 69: 1–21Google Scholar
  10. 10.
    Crawford GR, Wright PJ (1987) Characterization of novel viral polyproteins detected in cells infected by the flavivirus Kunjin and radiolabelled in the presence of the leucine analogue hydroxyleucine. J Gen Virol 68: 365–376Google Scholar
  11. 11.
    Deubel V, Kinney RM, Esposito JJ, Cropp CB, Vorndam AV, Monath TP, Trent DW (1988) Dengue 2 virus envelope protein expressed by a recombinant vaccinia virus fails to protect monkeys against dengue. J Gen Virol 69: 1921–1929Google Scholar
  12. 12.
    Falgout B, Chanock R, Lai C-J (1989) Proper processing of dengue virus nonstructural glycoprotein NS 1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a. J Virol 63: 1852–1860Google Scholar
  13. 13.
    Gruenberg A, Woo WS, Biedrzycka A, Wright PJ (1988) Partial nucleotide sequence of the structural protein of dengue type 2 New Guinea C and PUO-218 strains. J Gen Virol 69: 1391–1398Google Scholar
  14. 14.
    Hahn YS, Galler R, Hunkapillar T, Dalrymple JM, Strauss JH, Strauss EG (1988) Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses. Virology 162: 167–180Google Scholar
  15. 15.
    Hahn YS, Lenches EM, Galler R, Rice CM, Dalrymple J, Strauss JH (1990) Expression of the structural proteins of dengue 2 virus and yellow fever virus by recombinant vaccinia viruses. Arch Virol 115: 251–265Google Scholar
  16. 16.
    Irie K, Mohan PM, Sasguri Y, Putnak R, Padmanabhan R (1989) Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain). Gene 75: 197–211Google Scholar
  17. 17.
    Kozak M (1987) At least six nucleotides preceding the AUG initiation codon enhance translation in mammalian cells. J Mol Biol 196. 947–950Google Scholar
  18. 18.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227: 680–685Google Scholar
  19. 19.
    Mackett M, Smith GL, Moss B (1982) Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci USA 79: 7415–7419Google Scholar
  20. 20.
    Mackett M, Smith GL, Moss B (1984) General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol 49: 857–864Google Scholar
  21. 21.
    Markoff L (1989) In vitro processing of dengue virus structural proteins: cleavage of the pre-membrane protein. J Virol 63: 3345–3352Google Scholar
  22. 22.
    Mason PW, Pincus S, Fournier MJ, Mason TL, Shope RE, Paoletti E (1991) Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection. Virology 180: 294–305Google Scholar
  23. 23.
    Naeve CW, Trent DW (1978) Identification of Saint Louis encephalitis virus mRNA. J Virol 25: 535–545Google Scholar
  24. 24.
    Rhim JS, Cho HY, Hubner RJ (1975) Non-producer human cells induced by murine sarcoma virus. Int J Cancer 15: 23–29Google Scholar
  25. 25.
    Rice CM, Lenches EM, Eddy AR, Shin SJ, Sheets RL, Strauss JH (1985) Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science 229: 726–733Google Scholar
  26. 26.
    Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) Labelling DNA to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113: 237–251Google Scholar
  27. 27.
    Ruiz-Linares A, Cahour A, Deprès P, Girard M, Bouloy M (1989) Processing of yellow fever virus polyprotein: role of cellular proteases in maturation of the structural proteins. J Virol 63: 4199–4209Google Scholar
  28. 28.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467Google Scholar
  29. 29.
    Shapiro D, Brandt WE, Russell PK (1972) Change involving a viral membrane glycoprotein during morphogenesis of group B arboviruses. Virology 50: 906–911Google Scholar
  30. 30.
    Smith GW, Wright PJ (1985) Synthesis of proteins and glycoproteins in dengue type 2 virus-infected Vero and Aedes albopictus cells. J Gen Virol 66: 559–571Google Scholar
  31. 31.
    Speight G, Coia G, Parker MD, Westaway EG (1988) Gene mapping and positive identification of the non-structural proteins NS2A, NS2B, NS 3, NS4B, and NS 5 of the flavivirus Kunjin and their cleavage sites. J Gen Virol 69: 23–34Google Scholar
  32. 32.
    Speight G, Westaway EG (1989) Carboxy-terminal analysis of nine proteins specified by the flavivirus Kunjin: evidence that only the intracellular core protein is truncated. J Gen Virol 70: 2209–2214Google Scholar
  33. 33.
    Svitkin YV, Lyapustin VN, Lashkevich VA, Agol VI (1984) Differences between translation products of thick-borne encephalitis virus RNA in cell-free systems from Krebs-2 cell and rabbit reticulocytes: involvement of membranes in the processing of nascent precursors of flavivirus structural proteins. Virology 135: 536–541Google Scholar
  34. 34.
    von Heijne G (1985) Signal sequences: the limits of variation. J Mol Biol 184: 99–105Google Scholar
  35. 35.
    Wengler G, Castle E, Leidner U, Nowak T, Wengler G (1985) Sequence analysis of the membrane protein V 3 of the flavivirus West Nile virus and of its gene. Virology 147: 264–274Google Scholar
  36. 36.
    Westaway EG (1980) Replication of flaviviruses. In: Schlesinger RW (ed) The togaviruses: biology, structure, replication. Academic Press, New York, pp 531–581Google Scholar
  37. 37.
    Westaway EG, Brinton MA, Gaidamovich SYA, Horzinek MC, Igarashi A, Lvov DK, Porterfield JS, Russell PK, Trent DW (1985) Flaviviridae. Intervirology 24: 183–192Google Scholar
  38. 38.
    Wright PJ, Cauchi MR, Ng ML (1989) Definition of the carboxy-termini of the three glycoproteins specified by dengue virus type 2. Virology 171: 61–67Google Scholar
  39. 39.
    Zhao B, Prince G, Horswood R, Eckels K, Summers P, Chanock R, Lai C-J (1987) Expression of dengue virus structural proteins and nonstructural protein NSI by a recombinant vaccinia virus. J Virol 61: 4019–4022Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • A. Gruenberg
    • 1
  • P. J. Wright
    • 1
  1. 1.Department of MicrobiologyMonash UniversityClaytonAustralia

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