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. 1996 Sep;70(9):6340–6346. doi: 10.1128/jvi.70.9.6340-6346.1996

The UL97 gene product of human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase.

D Michel 1, I Pavić 1, A Zimmermann 1, E Haupt 1, K Wunderlich 1, M Heuschmid 1, T Mertens 1
PMCID: PMC190660  PMID: 8709262

Abstract

The temporal expression of the UL97 gene product during human cytomegalovirus (HCMV) infection of human foreskin fibroblasts (HFF) and subcellular localization of this protein were analyzed by using a polyclonal antiserum raised against a truncated UL97 protein of 47 kDa. The UL97 protein was detectable 16 h after infection by Western blot (immunoblot) analysis. Since only reduced UL97 expression occurred in the presence of two inhibitors of DNA replication, phosphonoacetic acid and ganciclovir, we conclude that UL97 is an early-late gene, requiring DNA replication for maximum expression. By indirect immunofluorescence, the protein could be visualized in the nuclei of virus-infected HFF 22 h after infection. Nuclear localization of the UL97 protein was also detected in thymidine kinase-deficient 143B cells infected with a recombinant vaccinia virus containing the entire UL97 open reading frame (ORF), as well as in HFF transiently expressing the entire UL97 ORF under the control of HCMV major immediate-early promoter. However, transiently expressed 5'-terminal deletion mutants of the UL97 ORF in addition showed a cytoplasmic localization of the UL97 protein, confirming the presence of a nuclear localization site in the N-terminal region of the protein. Our high-pressure liquid chromatography analyses confirmed the ganciclovir phosphorylation by the UL97 protein, but no specific phosphorylation of natural nucleosides was observed, indicating that the UL97 protein is not a nucleoside kinase. During plaque purification of recombinant UL97-deficient HCMV, this virus was growth defective; hence, we presume that UL97 may be essential for the viral life cycle.

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Selected References

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  1. Anderson D. J., Blobel G. Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 1983;96:111–120. doi: 10.1016/s0076-6879(83)96012-3. [DOI] [PubMed] [Google Scholar]
  2. Baldanti F., Silini E., Sarasini A., Talarico C. L., Stanat S. C., Biron K. K., Furione M., Bono F., Palù G., Gerna G. A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate. J Virol. 1995 Feb;69(2):796–800. doi: 10.1128/jvi.69.2.796-800.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chee M. S., Lawrence G. L., Barrell B. G. Alpha-, beta- and gammaherpesviruses encode a putative phosphotransferase. J Gen Virol. 1989 May;70(Pt 5):1151–1160. doi: 10.1099/0022-1317-70-5-1151. [DOI] [PubMed] [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. Analysis of the UL97 phosphotransferase coding sequence in clinical cytomegalovirus isolates and identification of mutations conferring ganciclovir resistance. J Infect Dis. 1995 Mar;171(3):576–583. doi: 10.1093/infdis/171.3.576. [DOI] [PubMed] [Google Scholar]
  5. Cochrane A. W., Perkins A., Rosen C. A. Identification of sequences important in the nucleolar localization of human immunodeficiency virus Rev: relevance of nucleolar localization to function. J Virol. 1990 Feb;64(2):881–885. doi: 10.1128/jvi.64.2.881-885.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coligan J. E., Gates F. T., 3rd, Kimball E. S., Maloy W. L. Radiochemical sequence analysis of biosynthetically labeled proteins. Methods Enzymol. 1983;91:413–434. doi: 10.1016/s0076-6879(83)91039-x. [DOI] [PubMed] [Google Scholar]
  7. Depto A. S., Stenberg R. M. Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products. J Virol. 1989 Mar;63(3):1232–1238. doi: 10.1128/jvi.63.3.1232-1238.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dingwall C., Laskey R. A. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. doi: 10.1146/annurev.cb.02.110186.002055. [DOI] [PubMed] [Google Scholar]
  9. Garcia-Bustos J., Heitman J., Hall M. N. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. doi: 10.1016/0304-4157(91)90013-m. [DOI] [PubMed] [Google Scholar]
  10. Greenspan D., Palese P., Krystal M. Two nuclear location signals in the influenza virus NS1 nonstructural protein. J Virol. 1988 Aug;62(8):3020–3026. doi: 10.1128/jvi.62.8.3020-3026.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haarr L., Flatmark T. Evidence that deletion of coding sequences in the 5' end of the thymidine kinase gene of herpes simplex virus type 1 affects the stability of the gene products. J Gen Virol. 1987 Nov;68(Pt 11):2817–2829. doi: 10.1099/0022-1317-68-11-2817. [DOI] [PubMed] [Google Scholar]
  12. Haarr L., Marsden H. S., Preston C. M., Smiley J. R., Summers W. C., Summers W. P. 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. 1985 Nov;56(2):512–519. doi: 10.1128/jvi.56.2.512-519.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  14. Kalderon D., Richardson W. D., Markham A. F., Smith A. E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. doi: 10.1038/311033a0. [DOI] [PubMed] [Google Scholar]
  15. Koyano S., Suzutani T., Yoshida I., Azuma M. Analysis of phosphorylation pathways of antiherpesvirus nucleosides by varicella-zoster virus-specific enzymes. Antimicrob Agents Chemother. 1996 Apr;40(4):920–923. doi: 10.1128/aac.40.4.920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Littler E., Stuart A. D., Chee M. S. Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir. Nature. 1992 Jul 9;358(6382):160–162. doi: 10.1038/358160a0. [DOI] [PubMed] [Google Scholar]
  18. Lurain N. S., Spafford L. E., Thompson K. D. Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir. J Virol. 1994 Jul;68(7):4427–4431. doi: 10.1128/jvi.68.7.4427-4431.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lurain N. S., Thompson K. D., Holmes E. W., Read G. S. Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents. J Virol. 1992 Dec;66(12):7146–7152. doi: 10.1128/jvi.66.12.7146-7152.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marsden H. S., Haarr L., Preston C. M. Processing of herpes simplex virus proteins and evidence that translation of thymidine kinase mRNA is initiated at three separate AUG codons. J Virol. 1983 May;46(2):434–445. doi: 10.1128/jvi.46.2.434-445.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Metzger C., Michel D., Schneider K., Lüske A., Schlicht H. J., Mertens T. Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus. J Virol. 1994 Dec;68(12):8423–8427. doi: 10.1128/jvi.68.12.8423-8427.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Richardson W. D., Mills A. D., Dilworth S. M., Laskey R. A., Dingwall C. Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell. 1988 Mar 11;52(5):655–664. doi: 10.1016/0092-8674(88)90403-5. [DOI] [PubMed] [Google Scholar]
  23. Richardson W. D., Roberts B. L., Smith A. E. Nuclear location signals in polyoma virus large-T. Cell. 1986 Jan 17;44(1):77–85. doi: 10.1016/0092-8674(86)90486-1. [DOI] [PubMed] [Google Scholar]
  24. Schmolke S., Drescher P., Jahn G., Plachter B. 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. J Virol. 1995 Feb;69(2):1071–1078. doi: 10.1128/jvi.69.2.1071-1078.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shiraki K., Ogino T., Yamanishi K., Takahashi M. Immunochemical characterization of pyrimidine kinase induced by varicella-zoster virus. J Gen Virol. 1985 Feb;66(Pt 2):221–229. doi: 10.1099/0022-1317-66-2-221. [DOI] [PubMed] [Google Scholar]
  26. Siomi H., Shida H., Nam S. H., Nosaka T., Maki M., Hatanaka M. Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell. 1988 Oct 21;55(2):197–209. doi: 10.1016/0092-8674(88)90043-8. [DOI] [PubMed] [Google Scholar]
  27. Stanat S. C., Reardon J. E., Erice A., Jordan M. C., Drew W. L., Biron K. K. Ganciclovir-resistant cytomegalovirus clinical isolates: mode of resistance to ganciclovir. Antimicrob Agents Chemother. 1991 Nov;35(11):2191–2197. doi: 10.1128/aac.35.11.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stinski M. F., Thomsen D. R., Stenberg R. M., Goldstein L. C. Organization and expression of the immediate early genes of human cytomegalovirus. J Virol. 1983 Apr;46(1):1–14. doi: 10.1128/jvi.46.1.1-14.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sullivan V., Talarico C. L., Stanat S. C., Davis M., Coen D. M., Biron K. K. A protein kinase homologue controls phosphorylation of ganciclovir in human cytomegalovirus-infected cells. Nature. 1992 Jul 9;358(6382):162–164. doi: 10.1038/358162a0. [DOI] [PubMed] [Google Scholar]
  30. Wathen M. W., Stinski M. F. Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection. J Virol. 1982 Feb;41(2):462–477. doi: 10.1128/jvi.41.2.462-477.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wing B. A., Huang E. S. Analysis and mapping of a family of 3'-coterminal transcripts containing coding sequences for human cytomegalovirus open reading frames UL93 through UL99. J Virol. 1995 Mar;69(3):1521–1531. doi: 10.1128/jvi.69.3.1521-1531.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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