1887

Abstract

Human cytomegalovirus encodes a number of phosphorylation-regulated proteins, including the autophosphorylating protein kinase pUL97 and the nuclear mRNA export factor pUL69. Recently, it was reported that the kinase inhibitor roscovitine induces an intranuclear aggregation of pUL69 in infected fibroblasts. Here, we demonstrate that pUL97-specific kinase inhibitors induce a similar pUL69 aggregation. Furthermore, a direct pUL69–pUL97 interaction was demonstrated by coimmunoprecipitation analyses. Deletion mapping identified the domains required for interaction in both proteins (1–140/478–532 in pUL69 and 231–336 in pUL97). Further analysis of the immunoprecipitates by kinase assays demonstrated the phosphorylation of pUL69 by pUL97. However, catalytically inactive mutants of pUL97 and interaction-negative fragments of pUL69 were phosphorylation-negative. Moreover, an analysis of the pUL69-mediated nuclear RNA export indicated a correlation of the export efficiency with the presence of active pUL97 kinase. These data suggest a specific pUL69–pUL97 interaction and pUL97-mediated phosphorylation which influences the regulatory activities of pUL69.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.005827-0
2009-03-01
2019-11-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/90/3/567.html?itemId=/content/journal/jgv/10.1099/vir.0.005827-0&mimeType=html&fmt=ahah

References

  1. Baek, M. C., Krosky, P. M., Pearson, A. & Coen, D. M. ( 2004; ). Phosphorylation of the RNA polymerase II carboxyl-terminal domain in human cytomegalovirus-infected cells and in vitro by the viral UL97 protein kinase. Virology 324, 184–193.[CrossRef]
    [Google Scholar]
  2. Biron, K. K., Harvey, R. J., Chamberlain, S. C., Good, S. S., Smith, A. A., III, Davis, M. G., Talarico, C. L., Miller, W. H., Ferris, R. & other authors ( 2002; ). Potent and selective inhibition of human cytomegalovirus replication by 1263W94, a benzimidazole l-riboside with a unique mode of action. Antimicrob Agents Chemother 46, 2365–2372.[CrossRef]
    [Google Scholar]
  3. Chee, M. S., Lawrence, G. L. & Barrell, B. G. ( 1989; ). Alpha-, beta- and gammaherpesviruses encode a putative phosphotransferase. J Gen Virol 70, 1151–1160.[CrossRef]
    [Google Scholar]
  4. Farjot, G., Buisson, M., Dodon, M. D., Gazzolo, L., Sergeant, A. & Mikaelian, I. ( 2000; ). Epstein–Barr virus EB2 protein exports unspliced RNA via a Crm-1-independent pathway. J Virol 74, 6068–6076.[CrossRef]
    [Google Scholar]
  5. Goldsmith, E. J., Akella, R., Min, X., Zhou, T. & Humphreys, J. M. ( 2007; ). Substrate and docking interactions in serine/threonine protein kinases. Chem Rev 107, 5065–5081.[CrossRef]
    [Google Scholar]
  6. Herget, T., Freitag, M., Morbitzer, M., Stamminger, T. & Marschall, M. ( 2004; ). A novel chemical class of pUL97 protein kinase-specific inhibitors with strong anti-cytomegaloviral activity. Antimicrob Agents Chemother 48, 4154–4162.[CrossRef]
    [Google Scholar]
  7. Hofmann, H., Floss, S. & Stamminger, T. ( 2000; ). Covalent modification of the transactivator protein IE2-p86 of human cytomegalovirus by conjugation to the ubiquitin-homologous proteins SUMO-1 and hSMT3b. J Virol 74, 2510–2524.[CrossRef]
    [Google Scholar]
  8. Hofmann, H., Sindre, H. & Stamminger, T. ( 2002; ). Functional interaction between the pp71 protein of human cytomegalovirus and the PML-interacting protein human Daxx. J Virol 76, 5769–5783.[CrossRef]
    [Google Scholar]
  9. Hume, A. J., Finkel, J. S., Kamil, J. P., Coen, D. M., Culbertson, M. R. & Kalejta, R. F. ( 2008; ). Phosphorylation of retinoblastoma protein by viral protein with cyclin-dependent kinase function. Science 320, 797–799.[CrossRef]
    [Google Scholar]
  10. Kamil, J. P. & Coen, D. M. ( 2007; ). Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65. J Virol 81, 10659–10668.[CrossRef]
    [Google Scholar]
  11. Krosky, P. M., Baek, M. C., Jahng, W. J., Barrera, I., Harvey, R. J., Biron, K. K., Coen, D. M. & Sethna, P. B. ( 2003; ). The human cytomegalovirus UL44 protein is a substrate for the UL97 protein kinase. J Virol 77, 7720–7727.[CrossRef]
    [Google Scholar]
  12. Lischka, P., Rosorius, O., Trommer, E. & Stamminger, T. ( 2001; ). A novel transferable nuclear export signal mediates CRM1-independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69. EMBO J 20, 7271–7283.[CrossRef]
    [Google Scholar]
  13. Lischka, P., Toth, Z., Thomas, M., Mueller, R. & Stamminger, T. ( 2006; ). The UL69 transactivator protein of human cytomegalovirus interacts with DEXD/H-Box RNA helicase UAP56 to promote cytoplasmic accumulation of unspliced RNA. Mol Cell Biol 26, 1631–1643.[CrossRef]
    [Google Scholar]
  14. Lischka, P., Thomas, M., Toth, Z., Mueller, R. & Stamminger, T. ( 2007; ). Multimerization of human cytomegalovirus regulatory protein UL69 via a domain that is conserved within its herpesvirus homologues. J Gen Virol 88, 405–410.[CrossRef]
    [Google Scholar]
  15. Lu, M. & Shenk, T. ( 1999; ). Human cytomegalovirus UL69 protein induces cells to accumulate in G1 phase of the cell cycle. J Virol 73, 676–683.
    [Google Scholar]
  16. Marschall, M., Freitag, M., Weiler, S., Sorg, G. & Stamminger, T. ( 2000; ). Recombinant GFP-expressing human cytomegalovirus as a tool for screening of antiviral agents. Antimicrob Agents Chemother 44, 1588–1597.[CrossRef]
    [Google Scholar]
  17. Marschall, M., Stein-Gerlach, M., Freitag, M., Kupfer, R., van den Bogaard, M. & Stamminger, T. ( 2001; ). Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97. J Gen Virol 82, 1439–1450.
    [Google Scholar]
  18. Marschall, M., Stein-Gerlach, M., Freitag, M., Kupfer, R., van den Bogaard, M. & Stamminger, T. ( 2002; ). Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle of antiviral therapy. J Gen Virol 83, 1013–1023.
    [Google Scholar]
  19. Marschall, M., Freitag, M., Suchy, P., Romaker, D., Kupfer, D., Hanke, M. & Stamminger, T. ( 2003; ). The protein kinase pUL97 of human cytomegalovirus interacts with and phosphorylates the DNA polymerase processivity factor pUL44. Virology 311, 60–71.[CrossRef]
    [Google Scholar]
  20. Marschall, M., Marzi, A., aus dem Siepen, P., Jochmann, R., Kalmer, M., Auerochs, S., Lischka, P., Leis, M. & Stamminger, T. ( 2005; ). Cellular p32 recruits cytomegalovirus kinase pUL97 to redistribute the nuclear lamina. J Biol Chem 280, 33357–33367.[CrossRef]
    [Google Scholar]
  21. Michel, D., Pavic, I., Zimmermann, A., Haupt, E., Wunderlich, K., Heuschmid, M. & Mertens, T. ( 1996; ). The UL97 gene product of human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase. J Virol 70, 6340–6346.
    [Google Scholar]
  22. Milbradt, J., Auerochs, S. & Marschall, M. ( 2007; ). Cytomegaloviral proteins pUL50 and pUL53 are associated with the nuclear lamina and interact with cellular protein kinase C. J Gen Virol 88, 2642–2650.[CrossRef]
    [Google Scholar]
  23. Milbradt, M., Auerochs, S., Sticht, H. & Marschall, M. ( 2009; ). Cytomegaloviral proteins that associate with the nuclear lamina: components of a postulated nuclear egress complex. J Gen Virol 90, 579–590.[CrossRef]
    [Google Scholar]
  24. Mocarski, E. S., Jr, Shenk, T. & Pass, R. F. ( 2007; ). Cytomegaloviruses. In Fields Virology, 5th edn, pp. 2701–2772. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott, Williams & Wilkins.
  25. Prichard, M. N., Gao, N., Jairath, S., Mulamba, G., Krosky, P., Coen, D. M., Parker, B. O. & Pari, G. S. ( 1999; ). A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency. J Virol 73, 5663–5670.
    [Google Scholar]
  26. Prichard, M. N., Sztul, E., Daily, S. L., Perry, A. L., Frederick, S. L., Gill, R. B., Hartline, C. B., Streblow, D. N., Varnum, S. M. & other authors ( 2008; ). Human cytomegalovirus UL97 kinase activity is required for the hyperphosphorylation of retinoblastoma protein and inhibits the formation of nuclear aggresomes. J Virol 82, 5054–5067.[CrossRef]
    [Google Scholar]
  27. Romaker, D., Schregel, V., Maurer, K., Auerochs, S., Marzi, A., Sticht, H. & Marschall, M. ( 2006; ). Analysis of the structure-activity relationship of four UL97 subfamily herpesviral protein kinases reveals partial but not full functional conservation. J Med Chem 49, 7044–7053.[CrossRef]
    [Google Scholar]
  28. Sanchez, V. & Spector, D. H. ( 2006; ). Cyclin-dependent kinase activity is required for efficient expression and posttranslational modification of human cytomegalovirus proteins and for production of extracellular particles. J Virol 80, 5886–5896.[CrossRef]
    [Google Scholar]
  29. Schleiss, M., Eickhoff, J., Auerochs, S., Leis, M., Abele, S., Rechter, S., Choi, Y., Anderson, J., Scott, G. & other authors ( 2008; ). Protein kinase inhibitors of the quinazoline class exert anti-cytomegaloviral activity in vitro and in vivo. Antiviral Res 79, 49–61.[CrossRef]
    [Google Scholar]
  30. Schregel, V., Auerochs, S., Jochmann, R., Maurer, K., Stamminger, T. & Marschall, M. ( 2007; ). Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97. J Gen Virol 88, 395–404.[CrossRef]
    [Google Scholar]
  31. Toth, Z., Lischka, P. & Stamminger, T. ( 2006; ). RNA-binding of the human cytomegalovirus transactivator protein UL69, mediated by arginine-rich motifs, is not required for nuclear export of unspliced RNA. Nucleic Acids Res 34, 1237–1249.[CrossRef]
    [Google Scholar]
  32. Winkler, M. & Stamminger, T. ( 1996; ). A specific subform of the human cytomegalovirus transactivator protein pUL69 is contained within the tegument of virus particles. J Virol 70, 8984–8987.
    [Google Scholar]
  33. Winkler, M., Rice, S. A. & Stamminger, T. ( 1994; ). UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression. J Virol 68, 3943–3954.
    [Google Scholar]
  34. Winkler, M., aus dem Siepen, T. & Stamminger, T. ( 2000; ). Functional interaction between pleiotropic transactivator pUL69 of human cytomegalovirus and the human homolog of yeast chromatin regulatory protein SPT6. J Virol 74, 8053–8064.[CrossRef]
    [Google Scholar]
  35. Wolf, D. G., Courcelle, C. T., Prichard, M. N. & Mocarski, E. S. ( 2001; ). Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation. Proc Natl Acad Sci U S A 98, 1895–1900.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.005827-0
Loading
/content/journal/jgv/10.1099/vir.0.005827-0
Loading

Data & Media loading...

Most Cited This Month

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