1887

Abstract

Alkhurma virus (ALKV) is a tick-borne class 4 flavivirus responsible for several human cases of haemorrhagic fever in Saudi Arabia, with no specific treatment currently available. The viral RNA encodes a serine protease (NS2B–NS3), essential for virus replication in infected cells, that constitutes an attractive target for antiviral compounds. In an attempt to identify residues and motifs on NS2B that are necessary for protease activity of the ALKV NS2B–NS3 complex, a series of modified NS2B–NS3 proteins was constructed, with point mutations on particular residues or with the NS2B domain derived from two different viruses. Four mutants and the two chimeric proteins exhibited reduction of protease activity against BAPNA (a -nitroanilide substrate). The results demonstrate that tight complementarity of the protein sequences is necessary for NS2B-dependent activation of NS3. The results also determine residues in the ALKV NS2B cofactor essential for protease activation, giving new insights into protease function in flaviviruses.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.82088-0
2006-11-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/87/11/3279.html?itemId=/content/journal/jgv/10.1099/vir.0.82088-0&mimeType=html&fmt=ahah

References

  1. 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]
  2. Bessaud M., Grard G., Peyrefitte C. N., Pastorino B., Rolland D., Charrel R. N., de Lamballerie X., Tolou H. J. 2005; Identification and enzymatic characterization of NS2B-NS3 protease of Alkhurma virus, a class-4 flavivirus. Virus Res 107:57–62 [CrossRef]
    [Google Scholar]
  3. Bessaud M., Pastorino B. A. M., Peyrefitte C. N., Rolland D., Grandadam M., Tolou H. J. 2006; Functional characterization of the NS2B/NS3 protease complex from seven viruses belonging to different groups inside the genus Flavivirus . Virus Res 120:79–90 [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., Yao N., Zhong W. & 10 other authors 2000; Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3. J Virol 74:4291–4301 [CrossRef]
    [Google Scholar]
  6. 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]
  7. Charrel R. N., Zaki A. M., Attoui H. & 7 other authors 2001; Complete coding sequence of the Alkhurma virus, a tick-borne flavivirus causing severe hemorrhagic fever in humans in Saudi Arabia. Biochem Biophys Res Commun 287:455–461 [CrossRef]
    [Google Scholar]
  8. 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]
  9. Erbel P., Schiering N., D'Arcy A. & 7 other authors 2006; Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. Nat Struct Mol Biol 13:372–373 [CrossRef]
    [Google Scholar]
  10. 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]
  11. 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 protease activity. J Virol 67:2034–2042
    [Google Scholar]
  12. 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]
  13. Ke S.-H., Madison E. L. 1997; Rapid and efficient site-directed mutagenesis by single-tube ‘megaprimer’ PCR method. Nucleic Acids Res 25:3371–3372 [CrossRef]
    [Google Scholar]
  14. Leung D., Schroder K., White H., Fang N.-X., Stoermer M. J., Abbenante G., Martin J. L., Young P. R., Fairlie D. P. 2001; Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors. J Biol Chem 276:45762–45771 [CrossRef]
    [Google Scholar]
  15. Niyomrattanakit P., Winoyanuwattikun P., Chanprapaph S., Angsuthanasombat C., Panyim S., Katzenmeier G. 2004; Identification of residues in the dengue virus type 2 cofactor that are critical for NS3 protease activation. J Virol 78:13708–13716 [CrossRef]
    [Google Scholar]
  16. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. 1997; The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882 [CrossRef]
    [Google Scholar]
  17. Wu C.-F., Wang S.-H., Sun C.-M., Hu S.-T., Syu W.-J. 2003; Activation of dengue protease autocleavage at the NS2B–NS3 junction by recombinant NS3 and GST–NS2B fusion proteins. J Virol Methods 114:45–54 [CrossRef]
    [Google Scholar]
  18. Zaki A. M. 1997; Isolation of a flavivirus related to the tick-borne encephalitis complex from human cases in Saudi Arabia. Trans R Soc Trop Med Hyg 91:179–181 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.82088-0
Loading
/content/journal/jgv/10.1099/vir.0.82088-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error