1887

Abstract

In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5′ fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protein and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs. The results indicated the presence of an N-terminal region within pUL97 which changed the intracellular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matrix fraction. Expression of all pUL97 mutants could be confirmed by Western blot analysis. pUL97-associated ganciclovir (GCV) phosphorylation in rVV-infected cells, determined quantitatively by HPLC analysis, was abolished completely using individual UL97 deletion mutants. Phosphorylation of full-length and some of the mutated pUL97 was detected in cells infected with the rVVs. The UL97 constructs carrying point mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V, and the 4 aa deletion 590AACR593, also resulted in decreased but not abolished phosphorylation of GCVinthe rVVsystem, whereas the phosphorylation of pUL97 itself was not influenced. The rVV system is a suitable method for quantitatively testing the functional relevance of pUL97 mutations.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-79-9-2105
1998-09-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/jgv/79/9/9747718.html?itemId=/content/journal/jgv/10.1099/0022-1317-79-9-2105&mimeType=html&fmt=ahah

References

  1. Baldanti F., Silini E., Sarasini A., Talarico C. L., Stanat S. C., Biron K. K., Furione M., Bono F., Palu G., Gerna G. 1995; A three- nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistanceof a human cytomegalovirus clinical isolate. Journal of Virology 69:796–800
    [Google Scholar]
  2. Chalfie M. T, Tu Y, Euskirchen G, Ward W. W, Prasher D. C. 1994; Green fluorescent protein as a marker for gene expression. Science 263:802–805
    [Google Scholar]
  3. Chee M. S., Lawrence G. L., Barrell B. G. 1989; Alpha-, beta- and gammaherpesviruses encodea putative phosphotransferase. Journal of General Virology 70:1151–1160
    [Google Scholar]
  4. Chou S, Erice A, Jordan M. C., Vercellotti G. M., Michels K. R., Talarico C. L., Stanat S. C., Biron K. K. 1995; Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance. Journal of Infectious Diseases 171:576–583
    [Google Scholar]
  5. Hanks S. K., Quinn A. M., Hunter T. 1988; The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241:42–52
    [Google Scholar]
  6. Hanson M, Preheim L. C., Chou S., Talarico C. L., Biron K. K., Erice A. 1995; Novel mutation in the UL97 gene of a clinical cytomegalovirus strain conferring resistance to ganciclovir. Antimicrobial Agents and Chemotherapy 39:1204–1205
    [Google Scholar]
  7. He Z., He Y., Kim Y., Chu L., Ohmstede C., Biron K. K., Coen D. M. 1997; The human cytomegalovirus UL97 protein is aprotein kinase that autophosphorylates on serines and threonines. Journal of Virology 71:405–411
    [Google Scholar]
  8. Kamps M. P., Taylor S. S., Sefton B. M. 1984; Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites. Nature 310:589–592
    [Google Scholar]
  9. Knighton D. R., Zheng J. H., Ten-Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. 1991; Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253:414–420
    [Google Scholar]
  10. Laemmli U. K. 1970; Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:681–685
    [Google Scholar]
  11. Landt O., Grunert H. -P., Hahn U. 1990; A general method for site- directed mutagenesis using the polymerase chain reaction. Gene 96:125–128
    [Google Scholar]
  12. Littler E., Stuart A. D., Chee M. S. 1992; Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir. Nature 358:160–162
    [Google Scholar]
  13. Lurain N. S., Spafford L. E., Thompson K. D. 1994; Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir. Journal of Virology 68:4427–4431
    [Google Scholar]
  14. Metzger C., Michel D., Schneider K., Lüske A., Schlicht H. -J., Mertens T. 1994; Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus. Journal of Virology 68:8423–8427
    [Google Scholar]
  15. Michel D., Pavić I., Zimmermann A., Haupt E., Wunderlich K., Heuschmid M., Mertens T. 1996; The UL97 gene product of the human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase. Journal of Virology 70:6340–6347
    [Google Scholar]
  16. Ng T. I., Talarico C., Burnette T. C., Biron K., Roizman B. 1996; Partial substitution of the functions of the herpes simplex virus 1 UL13 gene by the human cytomegalovirus UL97 gene. Virology 225:347–358
    [Google Scholar]
  17. Prasher D. C., Eckenrode V. K., Ward W. W., Prendergast F. G., Cornier M. J. 1992; Primary structure of the Aequorea victoria green- fluorescent protein. Gene 111:229–233
    [Google Scholar]
  18. Purves F. C., Roizman B. 1992; The UL13 gene of herpes simplex virus 1 encodes the functions for posttranslational processing associated with phosphorylation ofthe regulatory protein alpha 22. Proceedings ofthe National Academy of Sciences, USA 89:7310–7314
    [Google Scholar]
  19. Sambrook J., Maniatis T., Fritsch E. F. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  20. Schmolke S., Drescher P., Jahn G., Plachter B. 1996; Nuclear targeting of the tegument protein pp65 (UL83) of human cytomegalovirus : an unusual bipartite nuclear localization signal functions with other portions of the protein to mediate its efficient nuclear transport. Journal of Virology 69:1071–1078
    [Google Scholar]
  21. Sullivan V., Talarico C. L., Stanat S. C., Davis M., Coen D. M., Biron K. K. 1992; A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells. Nature 358:162–164
    [Google Scholar]
  22. vanZeijl M., Fairhurst J., Baum E. Z., Sun L., Jones T. R. 1997; The human cytomegalovirus UL97 protein is phosphorylated and a component of virions. Virology 231:72–80
    [Google Scholar]
  23. Wierenga R. K., Hol W. G. J. 1983; Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant. Nature 302:842–844
    [Google Scholar]
  24. Wolf D. G., Smith I. L., Lee D. J., Freeman W. R., Flores-Aguilar M., Spector S. A. 1995; Mutations in human cytomegalovirus UL97 gene confer clinical resistance to ganciclovir and can be detected directly in patient plasma. Journal of Clinical Investigation 95:257–263
    [Google Scholar]
  25. Zhao L. J., Padmanabhan R. 1988; Nuclear transport of adenovirus DNA polymerase is facilitated by interaction with preterminal protein. Cell 55:1005–1015
    [Google Scholar]
  26. Zimmermann A., Michel D., Pavić I., Hampl W., Lüske A., Neyts J., DeClercq E., Mertens T. 1997; Phosphorylation of aciclovir, ganciclovir, penciclovir and S2242 by the cytomegalovirus UL97 protein : a quantitative analysis using recombinant vaccinia virus. Antiviral Research 36:35–42
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-79-9-2105
Loading
/content/journal/jgv/10.1099/0022-1317-79-9-2105
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