Journal of General Virology, Vol 79, 2105-2112, Copyright © 1998 by Society for General Microbiology

ARTICLES

Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself

D Michel, P Schaarschmidt, K Wunderlich, M Heuschmid, L Simoncini, D Muhlberger, A Zimmermann, I Pavic and T Mertens
Abteilung Virologie der Universitat Ulm, Germany. detlef.michel@medizin.uni-ulm.de

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 GCV in the rVV system, 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.

This article has been cited by other articles:

				M. N. Prichard, W. J. Britt, S. L. Daily, C. B. Hartline, and E. R. Kern Human Cytomegalovirus UL97 Kinase Is Required for the Normal Intranuclear Distribution of pp65 and Virion Morphogenesis J. Virol., December 15, 2005; 79(24): 15494 - 15502. [Abstract] [Full Text] [PDF]

				S. Hantz, D. Michel, A.-M. Fillet, V. Guigonis, G. Champier, M.-C. Mazeron, A. Bensman, F. Denis, T. Mertens, A. Dehee, et al. Early Selection of a New UL97 Mutant with a Severe Defect of Ganciclovir Phosphorylation after Valaciclovir Prophylaxis and Short-Term Ganciclovir Therapy in a Renal Transplant Recipient Antimicrob. Agents Chemother., April 1, 2005; 49(4): 1580 - 1583. [Abstract] [Full Text] [PDF]

				D. Michel, I. Milotic, M. Wagner, B. Vaida, J. Holl, R. Ansorge, and T. Mertens The human cytomegalovirus UL78 gene is highly conserved among clinical isolates, but is dispensable for replication in fibroblasts and a renal artery organ-culture system J. Gen. Virol., February 1, 2005; 86(2): 297 - 306. [Abstract] [Full Text] [PDF]

				M.-C. Baek, P. M. Krosky, and D. M. Coen Relationship between Autophosphorylation and Phosphorylation of Exogenous Substrates by the Human Cytomegalovirus UL97 Protein Kinase J. Virol., October 25, 2002; 76(23): 11943 - 11952. [Abstract] [Full Text] [PDF]

				L. De Bolle, D. Michel, T. Mertens, C. Manichanh, H. Agut, E. De Clercq, and L. Naesens Role of the Human Herpesvirus 6 U69-Encoded Kinase in the Phosphorylation of Ganciclovir Mol. Pharmacol., September 1, 2002; 62(3): 714 - 721. [Abstract] [Full Text] [PDF]

				M. Marschall, M. Stein-Gerlach, M. Freitag, R. Kupfer, M. van den Bogaard, and T. Stamminger Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle for antiviral therapy J. Gen. Virol., May 1, 2002; 83(5): 1013 - 1023. [Abstract] [Full Text] [PDF]

				M. Marschall, M. Stein-Gerlach, M. Freitag, R. Kupfer, M. van den Bogaard, and T. Stamminger Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97 J. Gen. Virol., June 1, 2001; 82(6): 1439 - 1450. [Abstract] [Full Text]

				M. Wagner, D. Michel, P. Schaarschmidt, B. Vaida, S. Jonjic, M. Messerle, T. Mertens, and U. Koszinowski Comparison between Human Cytomegalovirus pUL97 and Murine Cytomegalovirus (MCMV) pM97 Expressed by MCMV and Vaccinia Virus: pM97 Does Not Confer Ganciclovir Sensitivity J. Virol., November 15, 2000; 74(22): 10729 - 10736. [Abstract] [Full Text]

				D. Michel, S. Kramer, S. Hohn, P. Schaarschmidt, K. Wunderlich, and T. Mertens Amino Acids of Conserved Kinase Motifs of Cytomegalovirus Protein UL97 Are Essential for Autophosphorylation J. Virol., October 1, 1999; 73(10): 8898 - 8901. [Abstract] [Full Text] [PDF]

				J. S. Cannon, F. Hamzeh, S. Moore, J. Nicholas, and R. F. Ambinder Human Herpesvirus 8-Encoded Thymidine Kinase and Phosphotransferase Homologues Confer Sensitivity to Ganciclovir J. Virol., June 1, 1999; 73(6): 4786 - 4793. [Abstract] [Full Text]