1887

Abstract

A series of 46 charged-to-alanine mutations in the yellow fever virus NS2B–NS3 protease, previously characterized in cell-free and transient cellular expression systems, was tested for their effects on virus recovery. Four distinct plaque phenotypes were observed in cell culture: parental plaque-size (13 mutants), reduced plaque-size (17 mutants), small plaque-size (8 mutants) and no plaque-formation (8 mutants). No mutants displayed any temperature sensitivity based on recovery of virus after RNA transfection at 32 versus 37 °C. Most small plaque-mutants were defective in growth efficiency compared with parental virus. However not all small plaque-mutants had defective 2B/3 cleavage, with some showing selective defects at other non-structural protein cleavage sites. Revertant viruses were recovered for six mutations that caused reduced plaque sizes. Same-site and second-site mutations occurred in NS2B, and one second-site mutation occurred in the NS3 protease domain. Some reversion mutations ameliorated defects in cleavage activity and plaque size caused by the original mutation. These data indicate that certain mutations that reduce NS2B–NS3 protease cleavage activity cause growth restriction of yellow fever virus in cell culture. However, for at least two mutations, processing defects other than impaired cleavage activity at the 2B/3 site may account for the mutant phenotype. The existence of reversion mutations primarily in NS2B rather than NS3, suggests that the protease domain is less tolerant of structural perturbation compared with the NS2B protein.

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2005-05-01
2024-03-28
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References

  1. Amberg S. M., Nestorowicz A., McCourt D. W., Rice C. M. 1994; NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies. J Virol 68:3794–3802
    [Google Scholar]
  2. Arias C. F., Preugschat F., Strauss J. H. 1993; Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology 193:888–899 [CrossRef]
    [Google Scholar]
  3. Bazan J. F., Fletterick R. J. 1989; Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses. Virology 171:637–639 [CrossRef]
    [Google Scholar]
  4. Brinkworth R. I., Fairlie D. P., Leung D., Young P. R. 1999; Homology model of the dengue 2 virus NS3 protease: putative interactions with both substrate and NS2B cofactor. J Gen Virol 80:1167–1177
    [Google Scholar]
  5. Butkiewicz N. J., Wendel M., Zhang R. 10 other authors 1996; Enhancement of hepatitis C virus NS3 proteinase activity by association with NS4A-specific synthetic peptides: identification of sequence and critical residues of NS4A for the cofactor activity. Virology 225:328–338 [CrossRef]
    [Google Scholar]
  6. Cahour A., Falgout B., Lai C.-J. 1992; Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease. J Virol 66:1535–1542
    [Google Scholar]
  7. Chambers T. J., McCourt D. W., Rice C. M. 1990a; Production of yellow fever virus proteins in infected cells: identification of discrete polyprotein species and analysis of cleavage kinetics using region-specific polyclonal antisera. Virology 177:159–174 [CrossRef]
    [Google Scholar]
  8. Chambers T. J., Weir R. C., Grakoui A., McCourt D. W., Bazan J. F., Fletterick R. J., Rice C. M. 1990b; 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 87:8898–8902 [CrossRef]
    [Google Scholar]
  9. Chambers T. J., Grakoui A., Rice C. M. 1991; 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 65:6042–6050
    [Google Scholar]
  10. Chambers T. J., Nestorowicz A., Amberg S. M., Rice C. M. 1993; Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation and viral replication. J Virol 67:6797–6807
    [Google Scholar]
  11. Chambers T. J., Nestorowicz A., Rice C. M. 1995; Mutagenesis of the yellow fever virus NS2B/3 cleavage site: determinants of cleavage site specificity and effects on polyprotein processing and viral replication. J Virol 69:1600–1605
    [Google Scholar]
  12. Droll D. A., Krishna-Murthy H. M., Chambers T. J. 2000; Yellow fever virus NS2B-NS3 protease: charged-to-alanine mutagenesis and deletion analysis define regions important for protease complex formation and function. Virology 275:335–347 [CrossRef]
    [Google Scholar]
  13. Falgout B., Markoff L. 1995; Evidence that flavivirus NS1-NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic reticulum. J Virol 69:7232–7243
    [Google Scholar]
  14. Falgout B., Pethel M., Zhang Y. M., Lai C.-J. 1991; Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 65:2467–2475
    [Google Scholar]
  15. Falgout B., Miller R. H., Lai C.-J. 1993; Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 proteinase activity. J Virol 67:2034–2042
    [Google Scholar]
  16. Gorbalenya A. E., Donchenko A. P., Koonin E. V., Blinov V. M. 1989; N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases. Nucleic Acids Res 17:3889–3897 [CrossRef]
    [Google Scholar]
  17. Kummerer B. M., Rice C. M. 2002; Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles. J Virol 76:4773–4784 [CrossRef]
    [Google Scholar]
  18. Lin C., Amberg S. M., Chambers T. J., Rice C. M. 1993a; 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 67:2327–2335
    [Google Scholar]
  19. Lin C., Chambers T. J., Rice C. M. 1993b; Mutagenesis of conserved residues at the yellow fever 3/4A and 4B/5 dibasic cleavage sites: effects on cleavage efficiency and polyprotein processing. Virology 192:596–604 [CrossRef]
    [Google Scholar]
  20. Lin C., Thomson J. A., Rice C. M. 1995; A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro. J Virol 69:4373–4380
    [Google Scholar]
  21. Lindenbach B. D., Rice C. M. 2001; Flaviviridae . In Fields Virology , 3rd edn. pp  991–1041 Edited by Knipe D. M., Howley P. M., Griffin D. E., Lamb R. A., Martin M. A., Roizman B., Straus S. E. Philadelphia: Lippincott;
    [Google Scholar]
  22. Lobigs M. 1992; 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 73:2305–2312 [CrossRef]
    [Google Scholar]
  23. Lobigs M. 1993; Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3. Proc Natl Acad Sci U S A 90:6218–6222 [CrossRef]
    [Google Scholar]
  24. Love R. A., Parge H. E., Wickersham J. A. 7 other authors 1998; The conformation of hepatitis C virus NS3 proteinase with and without NS4A: a structural basis for the activation of the enzyme by its cofactor. Clin Diagn Virol 19:151–156
    [Google Scholar]
  25. Matusan A. E., Pryor M. J., Davidson A. D., Wright P. J. 2001; Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication. J Virol 75:9633–9643 [CrossRef]
    [Google Scholar]
  26. Murthy H. K., Clum S., Padmanabhan R. 1999; Dengue virus NS3 serine protease. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects. J Biol Chem 274:5573–5580 [CrossRef]
    [Google Scholar]
  27. Nestorowicz A., Chambers T. J., Rice C. M. 1994; 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 199:114–123 [CrossRef]
    [Google Scholar]
  28. Preugschat F., Strauss J. H. 1991; Processing of nonstructural proteins NS4A and NS4B of dengue 2 virus in vitro and in vivo. Virology 185:689–697 [CrossRef]
    [Google Scholar]
  29. Preugschat F., Yao C.-W., Strauss J. H. 1990; In vitro processing of dengue 2 nonstructural proteins NS2A, NS2B, and NS3. J Virol 64:4364–4374
    [Google Scholar]
  30. Rice C. M., Grakoui A., Galler R., Chambers T. J. 1989; Transcription of infectious yellow fever virus RNA from full-length cDNA templates produced by in vitro ligation. New Biol 1:285–296
    [Google Scholar]
  31. Satoh S., Tanji Y., Hijikata M., Kimura K., Shimotohno K. 1995; The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3), is essential for stable complex formation with NS4A. J Virol 69:4255–4260
    [Google Scholar]
  32. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. 1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
    [Google Scholar]
  33. Teo K. F., Wright P. J. 1997; Internal proteolysis of the NS3 protein specified by dengue virus 2. J Gen Virol 78:337–341
    [Google Scholar]
  34. Wengler G., Czaya G., Farber P. M., Hegemann J. H. 1991; 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 72:851–858 [CrossRef]
    [Google Scholar]
  35. Yamshchikov V. F., Compans R. W. 1994; Processing of the intracellular form of the West Nile virus capsid protein by the viral NS2B-NS3 protease: an in vitro study. J Virol 68:5765–5771
    [Google Scholar]
  36. Yamshchikov V. F., Compans R. W. 1995; Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease. J Virol 69:1995–2003
    [Google Scholar]
  37. Yusof R., Clum S., Wetzel M., Murthy K. H. M., Padmanabhan R. 2000; Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro . J Biol Chem 275:9963–9969 [CrossRef]
    [Google Scholar]
  38. Zhang L., Mohan P. M., Padmanabhan R. 1992; Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5. J Virol 66:7549–7554
    [Google Scholar]
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