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. 1995 Apr;69(4):1995–2003. doi: 10.1128/jvi.69.4.1995-2003.1995

Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease.

V F Yamshchikov 1, R W Compans 1
PMCID: PMC188864  PMID: 7884844

Abstract

One of the late processing events in the flavivirus replication cycle involves cleavage of the intracellular form of the flavivirus capsid protein (Cint) to the mature virion form (Cvir) lacking the carboxy-terminal stretch of hydrophobic amino acids which serves as a signal peptide for the downstream prM protein. This cleavage event was hypothesized to be effected by a viral protease and to be associated with virion formation. We have proposed a model of flavivirus virion formation in which processing of the C-prM precursor at the upstream signalase site is upregulated by interaction of the NS2B part of the protease with the prM signal peptide or with an adjacent carboxy-terminal region of the capsid protein in the precursor, and processing of Cint by the NS2B-NS3 protease follows the signalase cleavage. Recently, an alternative hypothesis was proposed which suggests a reverse order of these two cleavage events, namely, that cleavage of the C-prM precursor by the NS2B-NS3 protease at the Cint-->Cvir dibasic cleavage site is a prerequisite for the subsequent signalase cleavage of the prM signal peptide. To distinguish between these alternative models, we prepared a series of expression cassettes carrying mutations at the Cint-->Cvir dibasic cleavage site and investigated the effects of these mutations on signalase processing of C-prM and on formation and secretion of prM-E heterodimers. For certain mutated C-prM precursors, namely, for those with Lys-->Gly disruption of the dibasic site, efficient formation of prM was observed upon expression from larger cassettes encoding the viral protease, despite the absence of processing at the Cint-->Cvir cleavage site. Surprisingly, formation and secretion of prM-E heterodimers accompanied by late cleavage of prM was also observed for these cassettes, with an efficiency comparable to that of the wild-type expression cassette. These observations contradict the model in which cleavage of the C-prM precursor at the Cint-->Cvir dibasic site is a prerequisite for signalase cleavage.

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Selected References

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  1. Amberg S. M., Nestorowicz A., McCourt D. W., Rice C. M. NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies. J Virol. 1994 Jun;68(6):3794–3802. doi: 10.1128/jvi.68.6.3794-3802.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arias C. F., Preugschat F., Strauss J. H. Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology. 1993 Apr;193(2):888–899. doi: 10.1006/viro.1993.1198. [DOI] [PubMed] [Google Scholar]
  3. Bazan J. F., Fletterick R. J. Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses. Virology. 1989 Aug;171(2):637–639. doi: 10.1016/0042-6822(89)90639-9. [DOI] [PubMed] [Google Scholar]
  4. Biedrzycka A., Cauchi M. R., Bartholomeusz A., Gorman J. J., Wright P. J. 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. 1987 May;68(Pt 5):1317–1326. doi: 10.1099/0022-1317-68-5-1317. [DOI] [PubMed] [Google Scholar]
  5. Castle E., Leidner U., Nowak T., Wengler G., Wengler G. Primary structure of the West Nile flavivirus genome region coding for all nonstructural proteins. Virology. 1986 Feb;149(1):10–26. doi: 10.1016/0042-6822(86)90082-6. [DOI] [PubMed] [Google Scholar]
  6. Castle E., Nowak T., Leidner U., Wengler G., Wengler G. Sequence analysis of the viral core protein and the membrane-associated proteins V1 and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins. Virology. 1985 Sep;145(2):227–236. doi: 10.1016/0042-6822(85)90156-4. [DOI] [PubMed] [Google Scholar]
  7. Chambers T. J., Grakoui A., Rice C. M. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites. J Virol. 1991 Nov;65(11):6042–6050. doi: 10.1128/jvi.65.11.6042-6050.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Chambers T. J., Nestorowicz A., Amberg S. M., Rice C. M. Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J Virol. 1993 Nov;67(11):6797–6807. doi: 10.1128/jvi.67.11.6797-6807.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chambers T. J., Weir R. C., Grakoui A., McCourt D. W., Bazan J. F., Fletterick R. J., Rice C. M. Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8898–8902. doi: 10.1073/pnas.87.22.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dalbey R. E., Von Heijne G. Signal peptidases in prokaryotes and eukaryotes--a new protease family. Trends Biochem Sci. 1992 Nov;17(11):474–478. doi: 10.1016/0968-0004(92)90492-r. [DOI] [PubMed] [Google Scholar]
  12. Falgout B., Chanock R., Lai C. J. Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a. J Virol. 1989 May;63(5):1852–1860. doi: 10.1128/jvi.63.5.1852-1860.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Falgout B., Miller R. H., Lai C. J. Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity. J Virol. 1993 Apr;67(4):2034–2042. doi: 10.1128/jvi.67.4.2034-2042.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Falgout B., Pethel M., Zhang Y. M., Lai C. J. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol. 1991 May;65(5):2467–2475. doi: 10.1128/jvi.65.5.2467-2475.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gorbalenya A. E., Donchenko A. P., Blinov V. M., Koonin E. V. Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold. FEBS Lett. 1989 Jan 30;243(2):103–114. doi: 10.1016/0014-5793(89)80109-7. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Hallenberger S., Bosch V., Angliker H., Shaw E., Klenk H. D., Garten W. Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160. Nature. 1992 Nov 26;360(6402):358–361. doi: 10.1038/360358a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  22. Lin C., Amberg S. M., Chambers T. J., Rice C. M. Cleavage at a novel site in the NS4A region by the yellow fever virus NS2B-3 proteinase is a prerequisite for processing at the downstream 4A/4B signalase site. J Virol. 1993 Apr;67(4):2327–2335. doi: 10.1128/jvi.67.4.2327-2335.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lin C., Chambers T. J., Rice C. M. Mutagenesis of conserved residues at the yellow fever virus 3/4A and 4B/5 dibasic cleavage sites: effects on cleavage efficiency and polyprotein processing. Virology. 1993 Feb;192(2):596–604. doi: 10.1006/viro.1993.1076. [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. Lobigs M. Proteolytic processing of a Murray Valley encephalitis virus non-structural polyprotein segment containing the viral proteinase: accumulation of a NS3-4A precursor which requires mature NS3 for efficient processing. J Gen Virol. 1992 Sep;73(Pt 9):2305–2312. doi: 10.1099/0022-1317-73-9-2305. [DOI] [PubMed] [Google Scholar]
  26. Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [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. Mattila P., Korpela J., Tenkanen T., Pitkänen K. Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 1991 Sep 25;19(18):4967–4973. doi: 10.1093/nar/19.18.4967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Murray J. M., Aaskov J. G., Wright P. J. Processing of the dengue virus type 2 proteins prM and C-prM. J Gen Virol. 1993 Feb;74(Pt 2):175–182. doi: 10.1099/0022-1317-74-2-175. [DOI] [PubMed] [Google Scholar]
  30. Nestorowicz A., Chambers T. J., Rice C. M. Mutagenesis of the yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing, viral replication, and evidence for alternative processing of the NS2A protein. Virology. 1994 Feb 15;199(1):114–123. doi: 10.1006/viro.1994.1103. [DOI] [PubMed] [Google Scholar]
  31. Nowak T., Färber P. M., Wengler G., Wengler G. Analyses of the terminal sequences of West Nile virus structural proteins and of the in vitro translation of these proteins allow the proposal of a complete scheme of the proteolytic cleavages involved in their synthesis. Virology. 1989 Apr;169(2):365–376. doi: 10.1016/0042-6822(89)90162-1. [DOI] [PubMed] [Google Scholar]
  32. Preugschat F., Strauss J. H. Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo. Virology. 1991 Dec;185(2):689–697. doi: 10.1016/0042-6822(91)90540-r. [DOI] [PubMed] [Google Scholar]
  33. Preugschat F., Yao C. W., Strauss J. H. In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3. J Virol. 1990 Sep;64(9):4364–4374. doi: 10.1128/jvi.64.9.4364-4374.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pugachev K. V., Nomokonova NYu, Morozova O. V., Pletnev A. G. A short form of the tick-borne encephalitis virus NS3 protein. FEBS Lett. 1992 Feb 3;297(1-2):67–69. doi: 10.1016/0014-5793(92)80329-f. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Rice C. M., Lenches E. M., Eddy S. R., Shin S. J., Sheets R. L., Strauss J. H. Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science. 1985 Aug 23;229(4715):726–733. doi: 10.1126/science.4023707. [DOI] [PubMed] [Google Scholar]
  37. Rose J. K., Buonocore L., Whitt M. A. A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques. 1991 Apr;10(4):520–525. [PubMed] [Google Scholar]
  38. Ruiz-Linares A., Cahour A., Després P., Girard M., Bouloy M. Processing of yellow fever virus polyprotein: role of cellular proteases in maturation of the structural proteins. J Virol. 1989 Oct;63(10):4199–4209. doi: 10.1128/jvi.63.10.4199-4209.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Speight G., Coia G., Parker M. D., Westaway E. G. Gene mapping and positive identification of the non-structural proteins NS2A, NS2B, NS3, NS4B and NS5 of the flavivirus Kunjin and their cleavage sites. J Gen Virol. 1988 Jan;69(Pt 1):23–34. doi: 10.1099/0022-1317-69-1-23. [DOI] [PubMed] [Google Scholar]
  40. Speight G., Westaway E. G. Carboxy-terminal analysis of nine proteins specified by the flavivirus Kunjin: evidence that only the intracellular core protein is truncated. J Gen Virol. 1989 Aug;70(Pt 8):2209–2214. doi: 10.1099/0022-1317-70-8-2209. [DOI] [PubMed] [Google Scholar]
  41. Stevens T. M., Schlesinger R. W. Studies on the nature of dengue viruses. I. Correlation of particle density, infectivity, and RNA content of type 2 virus. Virology. 1965 Sep;27(1):103–112. doi: 10.1016/0042-6822(65)90147-9. [DOI] [PubMed] [Google Scholar]
  42. Wengler G., Czaya G., Färber P. M., Hegemann J. H. In vitro synthesis of West Nile virus proteins indicates that the amino-terminal segment of the NS3 protein contains the active centre of the protease which cleaves the viral polyprotein after multiple basic amino acids. J Gen Virol. 1991 Apr;72(Pt 4):851–858. doi: 10.1099/0022-1317-72-4-851. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Wright P. J., Cauchi M. R., Ng M. L. Definition of the carboxy termini of the three glycoproteins specified by dengue virus type 2. Virology. 1989 Jul;171(1):61–67. doi: 10.1016/0042-6822(89)90510-2. [DOI] [PubMed] [Google Scholar]
  45. Yamshchikov V. F., Compans R. W. Generation of long flavivirus expression cassettes by in vivo recombination and transient dominant selection. Gene. 1994 Nov 18;149(2):193–201. doi: 10.1016/0378-1119(94)90150-3. [DOI] [PubMed] [Google Scholar]
  46. Yamshchikov V. F., Compans R. W. Processing of the intracellular form of the west Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study. J Virol. 1994 Sep;68(9):5765–5771. doi: 10.1128/jvi.68.9.5765-5771.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Zhang L., Padmanabhan R. Role of protein conformation in the processing of dengue virus type 2 nonstructural polyprotein precursor. Gene. 1993 Jul 30;129(2):197–205. doi: 10.1016/0378-1119(93)90269-9. [DOI] [PubMed] [Google Scholar]

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