Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle for antiviral therapy Free

Abstract

The protein kinase pUL97, encoded by human cytomegalovirus (HCMV), is an important determinant of virus replication. Recently, indolocarbazoles were identified as a class of substances that inhibit the pUL97 kinase activity . In parallel, it was shown that indolocarbazoles interfere with HCMV replication; however, the causal relationship between inhibition of pUL97 kinase activity and virus replication has not been clarified. Here evidence is provided that indolocarbazole-mediated inhibition of virus replication is a direct result of diminished pUL97 protein kinase activity. In cell culture infections, a strong and selective antiviral activity was measured with respect to several strains of HCMV in contrast with other related or non-related viruses. For fine quantification, recombinant HCMVs expressing green fluorescent protein were used, demonstrating the high sensitivity towards compounds NGIC-I and Gö6976. Interestingly, a ganciclovir-resistant virus mutant (UL97-M460I) showed increased sensitivity to both compounds. Supporting this concept, transfection experiments with cloned pUL97 revealed that ganciclovir-resistant mutants were characterized by reduced levels of autophosphorylation compared with wild-type and possessed particularly high sensitivity to indolocarbazoles. Moreover, the Epstein–Barr virus-encoded homologous kinase, BGLF4, which showed a similar pattern of autophosphorylation and ganciclovir phosphorylation activities, was not inhibited. Importantly, a cytomegalovirus deletion mutant, lacking a functional UL97 gene and showing a severe impairment of replication, was completely insensitive to indolocarbazoles. Thus, our findings indicate that a specific block in the activity of pUL97 is the critical step in indolocarbazole-mediated inhibition of virus replication and that pUL97 might be targeted very efficiently by a novel antiviral therapy.

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2002-05-01
2024-03-29
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References

  1. Anders D. G., McCue L. A. 1996; The human cytomegalovirus genes required for DNA synthesis. Intervirology 39:378–388
    [Google Scholar]
  2. Cha T.-A., Tom E., Kemble G. W., Duke G. M., Mocarski E. S., Spaete R. R. 1996; Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. Journal of Virology 70:78–83
    [Google Scholar]
  3. Chee M. S., Lawrence G. L., Barrell B. G. 1989; Alpha-, beta- and gammaherpesviruses encode a putative phophotransferase. Journal of General Microbiology 70:1151–1160
    [Google Scholar]
  4. Chen M.-R., Chang S.-J., Huang H., Chen J.-Y. 2000; A protein kinase associated with Epstein–Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro . Journal of Virology 74:3093–3104
    [Google Scholar]
  5. Chulay J., Biron K., Wang L., Underwood M., Chamberlain S., Frick L., Good S., Davis M., Harvey R., Townsend L., Drach J., Koszalka G. 1999; Development of novel benzimidazole riboside compounds for treatment of cytomegalovirus disease. Advances in Experimental Medicine and Biology 458:129–134
    [Google Scholar]
  6. Crumpacker C. 2001; Antiviral therapy. In Fields Virology pp 393–433 Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
    [Google Scholar]
  7. Curran M., Noble S. 2001; Valganciclovir. Drugs 61:1145–1150
    [Google Scholar]
  8. Davis M. G., Talarico C. L., Underwood M. R., Baldanti F., Biron K. K. 1998; Mapping of a mutation in human cytomegalovirus resistant to 1263W94. In 23rd International Herpesvirus Workshop, York, UK. abstract 314
    [Google Scholar]
  9. Efferth T., Marschall M., Wang X., Huong S.-M., Hauber I., Olbrich A., Kronschnabl M., Stamminger T., Huang E.-S. 2002; Antiviral activity of artesunate towards wild-type, recombinant and ganciclovir-resistant human cytomegalovirus. Journal of Medical Virology (in Press)
    [Google Scholar]
  10. Emery V. C., Griffiths P. D. 2000; Prediction of cytomegalovirus load and resistance patterns after antiviral chemotherapy. Proceedings of the National Academy of Sciences, USA 97:8039–8044
    [Google Scholar]
  11. Erice A. 1999; Resistance of human cytomegalovirus to antiviral drugs. Clinical Microbiology Reviews 12:286–297
    [Google Scholar]
  12. Goekjian P. G., Jirousek M. R. 1999; Protein kinase C in treatment of diseases: signal transduction pathways, inhibitors and agents in development. Current Medicinal Chemistry 6:877–903
    [Google Scholar]
  13. Gustafson E. A., Chillemi A. C., Sage D. R., Fingeroth J. D. 1998; The Epstein-Barr virus thymidine kinase does not phosphorylate ganciclovir or acyclovir and demonstrates a narrow substrate specificity compared to the herpes simplex virus type 1 thymidine kinase. Antimicrobial Agents and Chemotherapy 42:2923–2931
    [Google Scholar]
  14. He Z., He Y. S., Kim Y., Chu L., Ohmstede C., Biron K. K., Coen D. M. 1997; The human cytomegalovirus UL97 protein is a protein kinase that autophosphorylates on serines and threonines. Journal of Virology 71:405–411
    [Google Scholar]
  15. Jabs D. A., Martin B. K., Forman M. S., Dunn J. P., Davis J. L., Weinberg D. V., Biron K. K., Baldanti F. 2001; Mutations conferring ganciclovir resistance in a cohort of patients with acquired immunodeficiency syndrome and cytomegalovirus retinitis. Journal of Infectious Diseases 183:333–337
    [Google Scholar]
  16. Kawaguchi Y., Matsumura T., Roizman B., Hirai K. 1999; Cellular elongation factor 1δ is modified in cells infected with representative alpha-, beta-, or gammaherpesviruses. Journal of Virology 73:4456–4460
    [Google Scholar]
  17. Kleinschroth J., Hartenstein J., Rudolph C., Schächtele C. 1993; Non-glycosidic/non-aminoalkyl-substituted indolocarbazoles as inhibitors of protein kinase C. Bioorganic and Medicinal Chemistry Letters 3:1959–1964
    [Google Scholar]
  18. Krosky P. M., Baek M.-C., Barrera I., Harvey R. J., Biron K. K., Coen D. M., Sethna P. B. 1999; HCMV UL97 phosphorylates HCMV UL44 in vitro. In 24th International Herpesvirus Workshop, Boston, USA. abstract 12016
    [Google Scholar]
  19. Kunkel T. A. 1985; Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences, USA 82:488–492
    [Google Scholar]
  20. Limaye A. P., Corey L., Koelle D. M., Davis C. L., Boeckh M. 2000; Emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants. Lancet 356:645–649
    [Google Scholar]
  21. 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]
  22. Liu W., Shum C., Martin D. F., Kuppermann B. D., Hall A. J., Margolis T. P. 2000; Prevalence of antiviral drug resistance in untreated patients with cytomegalovirus retinitis. Journal of Infectious Diseases 182:1234–1238
    [Google Scholar]
  23. Lowance D., Neumayer H. H., Legendre C. M., Squifflet J.-P., Kovarik J., Brennan P. J., Norman D., Mendez R., Keating M. R., Coggon G. L., Crisp A., Lee I. C. 1999; Valacyclovir for the prevention of cytomegalovirus disease after renal transplantation. New England Journal of Medicine 340:1462–1470
    [Google Scholar]
  24. McSharry J. J., McDonough A., Olson B., Talarico C., Davis M., Biron K. K. 2001; Inhibition of ganciclovir-susceptible and -resistant human cytomegalovirus clinical isolates by the benzimidazole l-riboside 1263W94. Clinical and Diagnostic Laboratory Immunology 8:1279–1281
    [Google Scholar]
  25. 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. Antimicrobial Agents and Chemotherapy 44:1588–1597
    [Google Scholar]
  26. Marschall M., Stein-Gerlach M., Freitag M., Kupfer R., van den Bogaard M., Stamminger T. 2001a; Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97. Journal of General Virology 82:1439–1450
    [Google Scholar]
  27. Marschall M., Stein-Gerlach M., Freitag M., Kupfer R., van den Bogaard M., Stamminger T. 2001b; Inhibition of the pUL97 protein kinase is an antiviral strategy to interfere with the replication of human cytomegalovirus. In 26th International Herpesvirus Workshop. Regensburg, Germany. abstract 1029
    [Google Scholar]
  28. 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 nuclear localization but is not a nucleoside kinase. Journal of Virology 70:6340–6346
    [Google Scholar]
  29. Michel D., Schaarschmidt P., Wunderlich K., Heuschmid M., Simoncini L., Mühlberger D., Zimmermann A., Pavic I., Mertens T. 1998; Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself. Journal of General Virology 79:2105–2112
    [Google Scholar]
  30. Michel D., Kramer S., Höhn S., Schaarschmidt P., Wunderlich K., Mertens T. 1999; Amino acids of conserved kinase motifs of cytomegalovirus protein UL97 are essential for autophosphorylation. Journal of Virology 73:8898–8901
    [Google Scholar]
  31. 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]
  32. Pass R. F. 2001; Cytomegaloviruses. In Fields Virology pp 2675–2705 Edited by Knipe D. M., Howley P. M. Philadelphia: Lippincott Williams & Wilkins;
    [Google Scholar]
  33. Perry C. M., Balfour J. A. B. 1999; Fomivirsen. Drugs 57:375–380
    [Google Scholar]
  34. Pindur U., Kim Y. S., Mehrabani F. 1999; Advances in indolo(2,3-a)carbazole chemistry: design and synthesis of protein kinase C and topoisomerase I inhibitors. Current Medicinal Chemistry 6:29–69
    [Google Scholar]
  35. 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. Journal of Virology 73:5663–5670
    [Google Scholar]
  36. Prichard M. N., Penfold M. E., Duke G. M., Speate R. R., Kemble G. W. 2001; A review of genetic differences between limited and extensively passaged human cytomegalovirus strains. Reviews in Medical Virology 11:191–200
    [Google Scholar]
  37. Schmidt B., Walter H., Moschik B., Paatz C., van Vaerenbergh K., Vandamme A.-M., Schmitt M., Harrer T., Überla K., Korn K. 2000; Simple algorithm derived from a geno-/phenotypic database to predict HIV-1 protease inhibitor resistance. AIDS 14:1731–1738
    [Google Scholar]
  38. Scholz M., Doerr H. W., Cinatl J. 2001; Inhibition of cytomegalovirus immediate early gene expression: a therapeutic option?. Antiviral Research 49:129–145
    [Google Scholar]
  39. Slater M. J., Cockerill S., Baxter R., Bonser R. W., Gohil K., Gowrie C., Robinson J. E., Littler E., Parry N., Randall R., Snowden W. 1999; Indolocarbazoles: potent, selective inhibitors of human cytomegalovirus replication. Bioorganic and Medicinal Chemistry Letters 7:1067–1074
    [Google Scholar]
  40. 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]
  41. Talarico C. L., Burnette T. C., Miller W. H., Smith S. L., Davis M. G., Stanat S. C., Ng T. I., He Z., Coen D. M., Roizman B., Biron K. K. 1999; Acyclovir is phosphorylated by the human cytomegalovirus UL97 protein. Antimicrobial Agents and Chemotherapy 43:1941–1946
    [Google Scholar]
  42. 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. Proceedings of the National Academy of Sciences, USA 98:1895–1900
    [Google Scholar]
  43. Yao Z.-Q., Gallez-Hawkins G., Lomeli N. A., Li X., Molinder K. M., Diamond D. J., Zaia J. A. 2001; Site-directed mutation in a conserved kinase domain of human cytomegalovirus-pp65 with preservation of cytotoxic T lymphocyte targeting. Vaccine 19:1628–1635
    [Google Scholar]
  44. Zacny V., Gershburg E., Davis M. G., Biron K., Pagano J. S. 1999; Inhibition of Epstein-Barr virus replication by a benzimidazole l-riboside: novel antiviral mechanism of 5,6-dichloro-2-(isopropylamino)-1-β-l-ribofuranosyl-1H-benzimidazole. Journal of Virology 73:7271–7277
    [Google Scholar]
  45. Zimmermann A., Wilts H., Lenhardt M., Hahn M., Mertens T. 2000; Indolocarbazoles exhibit strong antiviral activity against human cytomegalovirus and are potent inhibitors of the pUL97 protein kinase. Antiviral Research 48:49–60
    [Google Scholar]
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