1887

Abstract

The intact virion of bluetongue virus comprises ten segments of dsRNA enclosed in two concentric protein capsids. The core, which is transcriptionally active, includes three minor proteins (VP1, VP4 and VP6) which are considered to be the candidates for the core-associated enzymes that transcribe and modify full-length mRNA copies for each of the ten genome segments. Using purified recombinant VP4 protein and core-like particles containing VP4, in this report it is demonstrated that VP4 has nucleoside triphosphatase (NTPase) activity. VP4 is a nonspecific NTPase that hydrolyses four types of ribo- nucleoside triphosphate (NTP) to the corresponding nucleoside diphosphate. The substrate preference was GTP ATP UTP CTP. NTP hydrolysis by VP4 was maximal when the Mg or Ca ion concentrations were 4 mM or 6 mM, respectively. The presence of single-stranded polynucleotides poly(A), poly(U) and poly(C) had little effect on the NTPase activity. Although the enzyme exhibited a broad temperature optimum around 40 °C, the pH optimum was sharp, between pH 7 5 and 8. The and of ATP hydrolysis were calculated to be 0·25 0·05 μM ATP and 55 4 pmol ATP hydrolysed min μg \ respectively. The K was affected by the addition of poly(A) to only a small extent in contrast to the V, which was increased by at least twofold.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-79-10-2475
1998-10-01
2022-08-14
Loading full text...

Full text loading...

/deliver/fulltext/jgv/79/10/9780054.html?itemId=/content/journal/jgv/10.1099/0022-1317-79-10-2475&mimeType=html&fmt=ahah

References

  1. Borsa J., Gover J., Chapman J. D. 1970; Presence of nucleoside triphosphate phosphohydrolase activity in purified virions of reovirus. Journal of Virology 6:295–302
    [Google Scholar]
  2. Chen S. Jr Toribara T. Y., Warner H. 1956; Microdetermination of phosphorus. Analytical Chemistry 28:1756–1758
    [Google Scholar]
  3. Costas J. M., Sutton G., Ramadevi N., Roy P. 1998; Guanylyltransferase and RNA 5ʹ-triphosphatase activities of the purified expressed VP4 protein of bluetongue virus : identification of the active nucleotidebinding site. Journal of Molecular Biology in press
    [Google Scholar]
  4. Eagles R. M., Balmori Melian E., Beck D. L., Gardner R. C., Forster R. L. 1994; Characterization of NTPase, RNA-binding and RNA- helicase activities of the cytoplasmic inclusion protein of tamarillo mosaic potyvirus. European Journal of Biochemistry 224:677–684
    [Google Scholar]
  5. French T. J., Roy P. 1990; Synthesis of bluetongue virus (BTV) core like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. Journal of Virology 64:1530–1536
    [Google Scholar]
  6. Gottlieb P., Strassman J., Mindich L. 1992; Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity. Journal of Virology 66:6220–6222
    [Google Scholar]
  7. Grifo J., Abramson R., Salter C., Merrick W. 1984; RNA- stimulated ATPase activity of eukaryotic initiation factors. Journal of Biological Chemistry 259:8648–8654
    [Google Scholar]
  8. Kapuler A. M., Mendelsohn N., Klett H., Acs G. 1970; Four base- specific nucleoside 5ʹ-triphosphatase in the subviral core of rep virus. Nature 225:1209–1213
    [Google Scholar]
  9. Kuo M.-D., Chin C., Hsu S.-L., Shiao J.-Y., Wang T.-M., Lin J.-H. 1996; Characterization of the NTPase activity of Japanese encephalitis virus NS3 protein. Journal of General Virology 77:2077–2084
    [Google Scholar]
  10. LeBlois H., French T., Mertens P. P., Burroughs J. N., Roy P. 1992; The expressed VP4 protein of bluetongue virus binds GTP and is the candidate guanylyl transferase of the virus. Virology 189:757–761
    [Google Scholar]
  11. Lowery C., Richardson J. 1977; Characterisation of the nucleoside triphosphate phosphohydrolase (ATPase) activity of RNA synthesis termination factor p.11. Influence of synthetic RNA homopolymers and random copolymers on the reaction. Journal of Biological Chemistry 25:1381–1385
    [Google Scholar]
  12. Noble S., Nibert M. L. 1997; Characterization of an ATPase activity in reovirus cores and its genetic association with core-shell protein lambdal. Journal of Virology 71:2182–2191
    [Google Scholar]
  13. Paatero A. O., Syvaoja J. E., Bamford D. H. 1995; Doublestranded RNA bacteriophage vphi-6 protein P4 is an unspecific nucleoside triphosphatase activated by calcium ions. Journal of Virology 69:6729–6734
    [Google Scholar]
  14. Paoletti E., Moss B. 1974; Two nucleic acid dependent nucleoside triphosphate phosphohydrases from vaccinia virus. Nucleoside substrate and polynucleotide cofactor specificities. Journal of Biological Chemistry 249:3281–3286
    [Google Scholar]
  15. Ramadevi N., Rodriguez J., Roy P. 1998; Leucine zipper-like domain is essential for dimerization and encapsidation of bluetongue virus nucleocapsid protein VP4. Journal of Virology 72:2983–2990
    [Google Scholar]
  16. Roy P., Marshall J. J. A., French J. J. 1990; Structure ofbluetongue virus genome and its encoded proteins. In Bluetongue Virus 162 pp 43–87 Roy P., Gorman B. M. Edited by Heidelberg: Springer-Verlag;
    [Google Scholar]
  17. Scheffner M., Knippers R., Stahl H. 1989; RNA unwinding activity of SV40 large T antigen. Cell 57:955–963
    [Google Scholar]
  18. Schwer B., Guthrie C. 1991; PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome (see comments). Nature 349:494–499
    [Google Scholar]
  19. Staueber N., Costas J. M., Sutton G., Monastyrskaya K., Roy P. 1997; Bluetong ue virus VP6 protein binds ATP and exhibits an RNA- dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates. Journal of Virology 71:7220–7226
    [Google Scholar]
  20. 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]
  21. Testa D., Banerjee A. 1979; Nucleoside diphosphate kinase activity in purified cores of vesicular stomatitis virus. Journal ofBiological Chemistry 254:9075–9079
    [Google Scholar]
  22. Urakawa T., Ritter G. D., Roy P. 1989; Expression of largest RNA segment and synthesis of VP1 protein of bluetongue virus in insect cells by recombinant baculovirus : association of VP1 protein with RNA polymerase activity. Nucleic Acids Research 17:7395–7401
    [Google Scholar]
  23. 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]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-79-10-2475
Loading
/content/journal/jgv/10.1099/0022-1317-79-10-2475
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