1887

Abstract

Japanese encephalitis (JE) virus NS3 protein and two N-terminally truncated (Δ1–148 and Δ1-323) forms of NS3 were engineered and expressed in as fusion proteins with a histidine tag at the N terminus. The purified recombinant proteins his-NS3 and his-NS3 were found to possess NTPase activity which was stimulated by single-stranded RNA, whereas NS3 did not. The requirements for MgCl and MnCl and the salt and pH ranges necessary for optimal activity of the enzyme were determined and shown to be slightly different from those of the NTPases of other flaviviruses. Poly(U) and poly(C) were better than poly(A) at stimulating the NTPase activities, in contrast to other flaviviral NTPases. The substrate preference was in the order GTP > ATP ≫ UTP > CTP. Interestingly, we found that Ca could not substitute for Mg; on the contrary, it inhibited NTPase activity. The removal of the N-terminal 148 amino acids enhanced NTPase activity, but further deletion of the region (amino acids 148–323) completely abolished the activity. Therefore, amino acids 148–323 contain a critical region required for NTPase activity.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-77-9-2077
1996-09-01
2021-10-24
Loading full text...

Full text loading...

/deliver/fulltext/jgv/77/9/JV0770092077.html?itemId=/content/journal/jgv/10.1099/0022-1317-77-9-2077&mimeType=html&fmt=ahah

References

  1. Arai N., Kornberg A. 1981; Rep protein as a helicase in an active, isolatable replication fork of duplex ϕ X174 DNA. Journal of Biological Chemistry 256:5294–5298
    [Google Scholar]
  2. Bartholomeusz A. I., Wright P. J. 1993; Synthesis of dengue virus RNA in vitro: initiation and the involvement of proteins NS3 and NS5. Archives of Virology 128:111–121
    [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
    [Google Scholar]
  4. Berridge M. J. 1993; Inositol triphosphate and calcium signalling. Nature 361:315–325
    [Google Scholar]
  5. Burke D. S., Leake C. J. 1986; Japanese encephalitis. In The Arboviruses: Epidemiology and Ecology vol 3 pp 64–92 Edited by Monath T. P. Boca Raton, Fla.: CRC Press;
    [Google Scholar]
  6. Chambers T. J., Weir R. C., Grakoui A., McCourt D. W., Bazan J. F., Fletterick R. J., Rice C. M. 1990; Evidence that the N-terminal domain of nonstructurai protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein. Proceedings of the National Academy of Sciences, USA 87:8898–8902
    [Google Scholar]
  7. Chu P. W. G., Westaway E. G. 1987; Characterisation of Kunjin virus RNA-dependent RNA polymerase: reinitiation of synthesis in vitro. Virology 157:330–337
    [Google Scholar]
  8. Clapham D. E. 1995; Calcium signaling. Cell 80:259–268
    [Google Scholar]
  9. Gorbalenya A. E., Donchenko A. P., Koonin E. V., Blinov V. M. 1989a; N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases. Nucleic Acids Research 17:3889–3897
    [Google Scholar]
  10. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. 1989b; Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Research 17:4713–4729
    [Google Scholar]
  11. Hashimoto H., Nomoto A., Watanabe K., Mori T., Takazawa T., Aizawa C., Kakagami T., Hiramatsu K. 1990; Molecular cloning and complete nucleotide sequence of the genome of Japanese encephalitis virus Beijing-1 strain. Virus Genes 1:305–317
    [Google Scholar]
  12. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157:105–132
    [Google Scholar]
  13. Lain S., Riechmann J. L., Martin M. T., Garcia J. A. 1989; Homologous poty virus and flavivirus proteins belonging to a superfamily of helicase-like proteins. Gene 82:357–362
    [Google Scholar]
  14. Lin J.-H., Chang M.-F., Baker S. C., Govindarajan S., Lai M. M. C. 1990; Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA. Journal of Virology 64:4051–4058
    [Google Scholar]
  15. Nitayaphan S., Grant J. A., Chang G. J., Trent E. W. 1990; Nucleotide sequence of the virulent SA-14 strain of Japanese encephalitis virus and its attenuated vaccine derivative, SA1414-2. Virology 177:541–552
    [Google Scholar]
  16. Pozzan T., Rizzuto R., Volpe P., Meldolesi J. 1994; Molecular and cellular physiology of intracellular calcium stores. Physiological Reviews 74:595–636
    [Google Scholar]
  17. Preugschat F., Lenches E. M., Strauss J. H. 1991; Flavivirus enzyme-substrate interactions studied with chimeric proteinases: identification of an intragenic locus important for substrate recognition. Journal of Virology 65:4749–4758
    [Google Scholar]
  18. Pugh B. F., Cox M. M. 1988; High salt activation of RecA protein ATPase in the absence of DNA. Journal of Biological Chemistry 263:76–83
    [Google Scholar]
  19. Rice C. M., Lenches E. M., Eddy S. R., Shin S. J., Sheets R. L., Strauss J. H. 1985; Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science 229:726–733
    [Google Scholar]
  20. Ruiz-Linares A., Cahour A., Despres P., Girard M., Bouloy J. 1989; Processing of yellow fever virus polyprotein: role of cellular proteases in maturation of the structural proteins. Journal of Virology 63:4199–4209
    [Google Scholar]
  21. Sumiyoshi H., Mori C., Fuke I., Morita K., Kuhara S., Kondou J., Kikuchi Y., Nagamatu H., Igarashi A. 1987; Complete nucleotide sequence of Japanese encephalitis virus genome RNA. Virology 161:497–510
    [Google Scholar]
  22. Suzich J. A., Tamura J. K., Palmer-Hill F., Warrener P., Grakoui A., Rice C. M., Feinstone S. M., Collett M. S. 1993; Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes. Journal of Virology 67:6152–6158
    [Google Scholar]
  23. Takegami T., Hotta S. 1989; In vitro synthesis of Japanese encephalitis virus (JEV) RNA: membrane and nuclear fractions of JEV-infected cells possess high levels of virus-specific RNA polymerase activity. Virus Research 13:337–350
    [Google Scholar]
  24. Takegami T., Sakamuro D., Furukawa T. 1994; Japanese encephalitis virus nonstructurai protein NS3 has RNA binding and ATPase activities. Virus Genes 9:105–112
    [Google Scholar]
  25. Tamura J. K., Warrener P., Collett M. S. 1993; RNA-stimulated NTPase activity associated with the p80 protein of the pestivirus bovine viral diarrhea virus. Virology 193:1–10
    [Google Scholar]
  26. Tufty R. M., Kretsinger R. H. 1975; Troponin and parvalbumin calcium binding regions predicted in myosin light chain and T4 lysozyme. Science 187:167–169
    [Google Scholar]
  27. Warrener P., Tamura J. K., Collett M. S. 1993; RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. Journal of Virology 67:989–996
    [Google Scholar]
  28. Wengler G., Wengler G. 1991; The carboxy-terminal part of the NS3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology 184:707–715
    [Google Scholar]
  29. Wengler G., Castle E., Leidner U., Nowak T., Wengler G. 1985; Sequence analysis of the membrane protein V3 of the flavivirus West Nile virus and of its gene. Virology 147:264–274
    [Google Scholar]
  30. 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. Journal of General Virology 72:851–858
    [Google Scholar]
  31. Yarranton G. T., Getter M. L. 1979; Enzyme-catalyzed DNA unwinding: studies on Escherichia coli Rep protein. Proceedings of the National Academy of Sciences, USA 76:1658–1662
    [Google Scholar]
  32. Zamenhof P. J., Villarejo M. 1972; Construction and properties of Escherichia coli strains exhibiting alpha-complementation of fi- galactosidase fragments in vivo . Journal of Bacteriology 110:171–178
    [Google Scholar]
  33. Zulkarnain E., Takegami T. 1993; Localization and functions of Japanese encephalitis virus nonstructurai proteins NS3 and NS5 for viral RNA synthesis in the infected cells. Microbiology and Immunology 37:239–243
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-77-9-2077
Loading
/content/journal/jgv/10.1099/0022-1317-77-9-2077
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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