Skip to content
1887

Journal of General Virology header logo Journal of General Virology

Volume 92, Issue 3

Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation No Access

Abstract

The pUL97 protein kinase encoded by human cytomegalovirus is a multifunctional determinant of the efficiency of viral replication and phosphorylates viral as well as cellular substrate proteins. Here, we report that pUL97 is expressed in two isoforms with molecular masses of approximately 90 and 100 kDa. ORF UL97 comprises an unusual coding strategy in that five in-frame ATG start codons are contained within the N-terminal 157 aa. Site-directed mutagenesis, transient expression of point and deletion mutants and proteomic analyses accumulated evidence that the formation of the large and small isoforms result from alternative initiation of translation, with the start points being at amino acids 1 and 74, respectively. kinase assays demonstrated that catalytic activity, in terms of autophosphorylation and histone substrate phosphorylation, was indistinguishable for the two isoforms. An analysis of the intracellular distribution of pUL97 by confocal laser-scanning microscopy demonstrated that both isoforms have a pronounced nuclear localization. Surprisingly, mapping experiments performed to identify the nuclear localization signal (NLS) of pUL97 strongly suggest that the mechanism of nuclear transport is distinct for the two isoforms. While the extreme N terminus (large isoform) comprises a highly efficient, bipartite NLS (amino acids 6–35), a second sequence apparently conferring a less efficient mode of nuclear translocation was identified downstream of amino acid 74 (small and large isoforms). Taken together, the findings argue for a complex mechanism of nuclear translocation for pUL97 which might be linked with fine-regulatory differences between the two isoforms.

  • Received:
  • Accepted:
  • Published Online:
© SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.026799-0
2011-03-01
2025-05-14
Loading full text...

Full text loading...

References

  1. Azzeh, M., Honigman, A., Taraboulos, A., Rouvinski, A. & Wolf, D. G.(2006). Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity. Virology 354, 69–79.[CrossRef] [Google Scholar]
  2. 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]
  3. Becke, S., Fabre-Mersseman, V., Aue, S., Auerochs, S., Sedmak, T., Wolfrum, U., Strand, D., Marschall, M., Plachter, B. & Reyda, S.(2010). Modification of the major tegument protein pp65 of 1 human cytomegalovirus inhibits viral growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells. J Gen Virol 91, 2531–2541.[CrossRef] [Google Scholar]
  4. 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]
  5. Combet, C., Blanchet, C., Geourjon, C. & Deléage, G.(2000). NPS@: network protein sequence analysis. Trends Biochem Sci 25, 147–150.[CrossRef] [Google Scholar]
  6. Conti, E., Uy, M., Leighton, L., Blobel, G. & Kuriyan, J.(1998). Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin α. Cell 94, 193–204.[CrossRef] [Google Scholar]
  7. Cunningham, C., Gatherer, D., Hilfrich, B., Baluchova, K., Dargan, D. J., Thomson, M., Griffiths, P. D., Wilkinson, G. W., Schulz, T. F. & Davison, A. J.(2010). Sequences of complete human cytomegalovirus genomes from infected cell cultures and clinical specimens. J Gen Virol 91, 605–615.[CrossRef] [Google Scholar]
  8. Degrève, B., Johansson, M., De Clercq, E., Karlsson, A. & Balzarini, J.(1998). Differential intracellular compartmentalization of herpetic thymidine kinases (TKs) in TK gene-transfected tumor cells: molecular characterization of the nuclear localization signal of herpes simplex virus type 1 TK. J Virol 72, 9535–9543. [Google Scholar]
  9. Degrève, B., Esnouf, R., De Clercq, E. & Balzarini, J.(1999). Characterization of multiple nuclear localization signals in herpes simlex virus type 1 thymidine kinase. Biochem Biophys Res Commun 264, 338–342.[CrossRef] [Google Scholar]
  10. Dinkel, H. & Sticht, H.(2007). A computational strategy for the prediction of functional linear peptide motifs in proteins. Bioinformatics 23, 3297–3303.[CrossRef] [Google Scholar]
  11. Fontes, M. R., The, T., Jans, D., Brinkworth, R. I. & Kobe, B.(2003). Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-α. J Biol Chem 278, 27981–27987.[CrossRef] [Google Scholar]
  12. Gill, R. B., Frederick, S. L., Hartline, C. B., Chou, S. & Prichard, M. N.(2009). Conserved retinoblastoma protein-binding motif in human cytomegalovirus UL97 kinase minimally impacts viral replication but affects susceptibility to maribavir. Virol J 6, 9.[CrossRef] [Google Scholar]
  13. Görlich, D., Prehn, S., Laskey, R. A. & Hartmann, E.(1994). Isolation of a protein that is essential for the first step of nuclear protein import. Cell 79, 767–778.[CrossRef] [Google Scholar]
  14. Görlich, D., Henklein, P., Laskey, R. A. & Hartmann, E.(1996). A 41 amino acid motif in importin-α confers binding to importin-β and hence transit into the nucleus. EMBO J 15, 1810–1817. [Google Scholar]
  15. Haarr, L., Marsden, H. S., Preston, C. M., Smiley, J. R., Summers, W. C. & Summers, W. P.(1985). Utilization of internal AUG codons for initiation of protein synthesis directed by mRNAs from normal and mutant genes encoding herpes simplex virus-specified thymidine kinase. J Virol 56, 512–519. [Google Scholar]
  16. Hamirally, S., Kamil, J. P., Ndassa-Colday, Y. M., Lin, A. J., Jahng, W. J., Baek, M. C., Noton, S., Silva, L. A., Simpson-Holley, M. & other authors(2009). Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress. PLoS Pathog 5, e1000275.[CrossRef] [Google Scholar]
  17. 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]
  18. Herold, A., Truant, R., Wiegand, H. & Cullen, B. R.(1998). Determination of the functional domain organization of the importin α nuclear import factor. J Cell Biol 143, 309–318.[CrossRef] [Google Scholar]
  19. 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]
  20. Kawaguchi, Y., Matsumura, T., Roizman, B. & Hirai, K.(1999). Cellular elongation factor 1δ is modified in cells infected with representative alpha-, beta-, or gammaherpesviruses. J Virol 73, 4456–4460. [Google Scholar]
  21. Kosugi, S., Hasebe, M., Tomita, M. & Yanagawa, H.(2009). Systematic identification of yeast cell cycle-dependent nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc Natl Acad Sci U S A 106, 10171–10176.[CrossRef] [Google Scholar]
  22. 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]
  23. Kunkel, T. A.(1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82, 488–492.[CrossRef] [Google Scholar]
  24. Lee, C. P. & Chen, M. R.(2010). Escape of herpesviruses from the nucleus. Rev Med Virol 20, 214–230.[CrossRef] [Google Scholar]
  25. Linding, R., Russell, R. B., Neduva, V. & Gibson, T. J.(2003). GlobPlot: exploring protein sequences for globularity and disorder. Nucleic Acids Res 31, 3701–3708.[CrossRef] [Google Scholar]
  26. Lischka, P., Sorg, G., Kann, M., Winkler, M. & Stamminger, T.(2003). A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin α/β pathway. J Virol 77, 3734–3748.[CrossRef] [Google Scholar]
  27. Marschall, M., Freitag, M., Weiler, S., Sorg, G. & Stamminger, T.(2000). Recombinant green fluorescent protein-expressing human cytomegalovirus as a tool for screening antiviral agents. Antimicrob Agents Chemother 44, 1588–1597.[CrossRef] [Google Scholar]
  28. 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]
  29. 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]
  30. Marschall, M., Freitag, M., Suchy, P., Romaker, D., Kupfer, R., 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]
  31. 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]
  32. Marsden, H. S., Haarr, L. & Preston, C. M.(1983). Processing of herpes simplex virus proteins and evidence that translation of thymidine kinase mRNA is initiated at three separate AUG codons. J Virol 46, 434–445. [Google Scholar]
  33. Michel, D., Papic, 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]
  34. Michel, D., Schaarschmidt, P., Wunderlich, K., Heuschmid, M., Simoncini, L., Mühlberger, D., Zimmermann, A., Papic, I. & Mertens, T.(1998). Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself. J Gen Virol 79, 2105–2112. [Google Scholar]
  35. Milbradt, J., 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]
  36. Milbradt, J., Webel, R., Auerochs, S., Sticht, H. & Marschall, M.(2010). Novel mode of phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus. J Biol Chem 285, 13979–13989.[CrossRef] [Google Scholar]
  37. Mocarski, E. S., Shenk, T. & Pass, R. F.(2007). Cytomegaloviruses. In Fields Virology, 5th edn, pp. 2701–2772. Edited by Knipe, D. M., Howley, P. M., Griffin, D. E., Lamb, R. A., Martin, M. A., Roizman, B. & Straus, S. E.. Philadelphia, PA. : Lippincott Williams & Wilkins. [Google Scholar]
  38. Moroianu, J., Blobel, G. & Radu, A.(1995). Previously identified protein of uncertain function is karyopherin α and together with karyopherin β docks import substrate at nuclear pore complexes. Proc Natl Acad Sci U S A 92, 2008–2011.[CrossRef] [Google Scholar]
  39. Prichard, M. N.(2009). Function of human cytomegalovirus UL97 kinase in viral infection and its inhibition by maribavir. Rev Med Virol 19, 215–229.[CrossRef] [Google Scholar]
  40. 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]
  41. Prichard, M. N., Britt, W. J., Daily, S. L., Hartline, C. B. & Kern, E. R.(2005). Human cytomegalovirus UL97 kinase is required for the normal intranuclear distribution of pp65 and virion morphogenesis. J Virol 79, 15494–15502.[CrossRef] [Google Scholar]
  42. 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]
  43. Rechter, S., Scott, G. M., Eickhoff, J., Zielke, K., Auerochs, S., Müller, R., Stamminger, T., Rawlinson, W. D. & Marschall, M.(2009). Cyclin-dependent kinases phosphorylate the cytomegalovirus RNA export protein pUL69 and modulate its nuclear localization and activity. J Biol Chem 284, 8605–8613.[CrossRef] [Google Scholar]
  44. Romaker, D., Schregel, V., Maurer, K., Auerochs, S., Marzi, A., Sticht, H. & Marschall, M.(2006). Analysis of the structure–activity relationship of four herpesviral UL97 subfamily protein kinases reveals partial but not full functional conservation. J Med Chem 49, 7044–7053.[CrossRef] [Google Scholar]
  45. 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]
  46. Sinzger, C., Hahn, G., Digel, M., Katona, R., Laib Sampaio, K., Messerle, M., Hengel, H., Koszinowski, U., Brune, W. & Adler, B.(2008). Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E. J Gen Virol 89, 359–368.[CrossRef] [Google Scholar]
  47. Sorg, G. & Stamminger, T.(1999). Mapping of nuclear localization signals by simultaneous fusion to green fluorescent protein and to β-galactosidase. Biotechniques 26, 858–862. [Google Scholar]
  48. Thomas, M., Rechter, S., Milbradt, J., Auerochs, S., Müller, R., Stamminger, T. & Marschall, M.(2009). Cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity. J Gen Virol 90, 567–578.[CrossRef] [Google Scholar]
  49. Truant, R. & Cullen, B. R.(1999). The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin β-dependent nuclear localization signals. Mol Cell Biol 19, 1210–1217. [Google Scholar]
  50. van Zeijl, 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.[CrossRef] [Google Scholar]
  51. Wing, B. A. & Huang, E. S.(1995). Analysis and mapping of a family of 3′-coterminal transcripts containing coding sequences for human cytomegalovirus open reading frames UL93 through UL99. J Virol 69, 1521–1531. [Google Scholar]
  52. Wolf, D. G., Honigman, A., Lazarovits, J., Tavor, E. & Panet, A.(1998). Characterization of the human cytomegalovirus UL97 gene product as a virion-associated protein kinase. Arch Virol 143, 1223–1232.[CrossRef] [Google Scholar]
  53. 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]
/content/journal/jgv/10.1099/vir.0.026799-0
Loading
Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation
J. Gen. Virol. 92, 638 (2011); https://doi.org/10.1099/vir.0.026799-0
/content/journal/jgv/10.1099/vir.0.026799-0
/content/journal/jgv/10.1099/vir.0.026799-0
Loading

Data & Media loading...

Supplements

vol. , part 3, pp. 638–649

Multiple sequence alignment. pUL97 sequences of human and simian cytomegaloviruses were analysed by using Tcoffee multiple sequence alignment tools.

Mass spectrometry analysis of the small isoform of pUL97.

Wild-type pUL97 showing nuclear localization as indistinguishable from the two individually expressed isoforms.

Confirmation of a functional NLS in amino acid region 6–35 of pUL97.

Oligonucleotide primers used in this study

[ Single PDF file] (210 KB)



PDF

Most cited 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