1887

Abstract

Human cytomegalovirus (HCMV) encodes a protein related to the large (R1) subunit of ribonucleotide reductase (RR), but does not encode the corresponding small (R2) subunit. The R1 homologue, UL45, lacks many catalytic residues, and its impact on deoxyribonucleotide (dNTP) production remains unknown. Here, UL45 is shown to accumulate at late stages of infection and to be a virion tegument protein. To study UL45 function in its genome context, UL45 was disrupted by transposon insertion. The UL45-knockout (UL45-KO) mutant exhibited a growth defect in fibroblasts at a low m.o.i. and also a cell-to-cell spread defect. This did not result from a reduced dNTP supply because dNTP pools were unchanged in resting cells infected with the mutant virus. Irrespective of UL45 expression, all cellular RR subunits – S-phase RR subunits, and the p53-dependent p53R2 – were induced by infection. p53R2 was targeted to the infected cell nucleus, suggesting that HCMV diverts a mechanism normally activated by DNA damage response. Cells infected with the UL45-KO mutant were moderately sensitized to Fas-induced apoptosis relative to those infected with the parental virus. Together with the report on the UL45-KO endotheliotropic HCMV mutant ( Hahn ., , 9551–9555, 2002 ), these data suggest that UL45 does not share the prominent antiapototic role attributed to the mouse cytomegalovirus homologue M45 ( Brune ., , 303–305, 2001 ).

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.19452-0
2003-12-01
2024-11-05
Loading full text...

Full text loading...

/deliver/fulltext/jgv/84/12/vir843359.html?itemId=/content/journal/jgv/10.1099/vir.0.19452-0&mimeType=html&fmt=ahah

References

  1. Aurelian L. 1998; Herpes simplex virus type 2: unique biological properties include neoplastic potential mediated by the PK domain of the large subunit of ribonucleotide reductase. Front Biosci 3:D237–249
    [Google Scholar]
  2. Biron K. K., Fyfe J. A., Stanat S. C., Leslie L. K., Sorrell J. B., Lambe C. U., Coen D. M. 1986; A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate. Proc Natl Acad Sci U S A 83:8769–8773
    [Google Scholar]
  3. Borst E. M., Hahn G., Koszinowski U. H., Messerle M. 1999; Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli : a new approach for construction of HCMV mutants. J Virol 73:8320–8329
    [Google Scholar]
  4. Britt W. J., Alford C. A. 1996; Cytomegalovirus. In Fields Virology , 3rd edn. vol 2 pp  2493–2523 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
    [Google Scholar]
  5. Brune W., Menard C., Heesemann J., Koszinowski U. H. 2001; A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism. Science 291:303–305
    [Google Scholar]
  6. Chabes A., Thelander L. 2000; Controlled protein degradation regulates ribonucleotide reductase activity in proliferating mammalian cells during the normal cell cycle and in response to DNA damage and replication blocks. J Biol Chem 275:17747–17753
    [Google Scholar]
  7. Chee M. S., Bankier A. T., Beck S. 12 other authors 1990; Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154:125–169
    [Google Scholar]
  8. Chung T. D., Wymer J. P., Smith C. C., Kulka M., Aurelian L. 1989; Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). J Virol 63:3389–3398
    [Google Scholar]
  9. Conner J. 1999; The unique N terminus of herpes simplex virus type 1 ribonucleotide reductase large subunit is phosphorylated by casein kinase 2, which may have a homologue in Escherichia coli . J Gen Virol 80:1471–1476
    [Google Scholar]
  10. Cooper J., Conner J., Clements J. B. 1995; Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase. J Virol 69:4979–4985
    [Google Scholar]
  11. Davis R., Thelander M., Mann G. J., Behravan G., Soucy F., Beaulieu P., Lavallee P., Graslund A., Thelander L. 1994; Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit. J Biol Chem 269:23171–23176
    [Google Scholar]
  12. Depto A. S., Stenberg R. M. 1992; Functional analysis of the true late human cytomegalovirus pp28 upstream promoter: cis -acting elements and viral trans -acting proteins necessary for promoter activation. J Virol 66:3241–3246
    [Google Scholar]
  13. Engstrom Y., Eriksson S., Jildevik I., Skog S., Thelander L., Tribukait B. 1985; Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits.. J Biol Chem 260:9114–9116
    [Google Scholar]
  14. Eriksson M., Uhlin U., Ramaswamy S., Ekberg M., Regnstrom K., Sjoberg B. M., Eklund H. 1997; Binding of allosteric effectors to ribonucleotide reductase protein R1: reduction of active-site cysteines promotes substrate binding. Structure 5:1077–1092
    [Google Scholar]
  15. Flemington E. K. 2001; Herpesvirus lytic replication and the cell cycle: arresting new developments. J Virol 75:4475–4481
    [Google Scholar]
  16. Fortunato E. A., McElroy A. K., Sanchez I., Spector D. H. 2000; Exploitation of cellular signaling and regulatory pathways by human cytomegalovirus. Trends Microbiol 8:111–119
    [Google Scholar]
  17. Gallina A., Simoncini L., Garbelli S. 7 other authors 1999; Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein. J Virol 73:1468–1478
    [Google Scholar]
  18. Gavrieli Y., Sherman Y., Ben-Sasson S. A. 1992; Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501
    [Google Scholar]
  19. Gerna G., Percivalle E., Baldanti F., Sozzani S., Lanzarini P., Genini E., Lilleri D., Revello M. G. 2000; Human cytomegalovirus replicates abortively in polymorphonuclear leukocytes after transfer from infected endothelial cells via transient microfusion events. J Virol 74:5629–5638
    [Google Scholar]
  20. Goldmacher V. S., Bartle L. M., Skaletskaya A. 10 other authors 1999; A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc Natl Acad Sci U S A 96:12536–12541
    [Google Scholar]
  21. Guittet O., Hakansson P., Voevodskaya N., Fridd S., Graslund A., Arakawa H., Nakamura Y., Thelander L. 2001; Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells. J Biol Chem 276:40647–40651
    [Google Scholar]
  22. Hahn G., Jores R., Mocarski E. S. 1998; Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc Natl Acad Sci U S A 95:3937–3942
    [Google Scholar]
  23. Hahn G., Khan H., Baldanti F., Koszinowski U. H., Revello M. G., Gerna G. 2002; The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics. J Virol 76:9551–9555
    [Google Scholar]
  24. Heineman T. C., Cohen J. I. 1994; Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro . J Virol 68:3317–3323
    [Google Scholar]
  25. Hobom U., Brune W., Messerle M., Hahn G., Koszinowski U. H. 2000; Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes. J Virol 74:7720–7729
    [Google Scholar]
  26. Idowu A. D., Fraser-Smith E. B., Poffenberger K. L., Herman R. C. 1992; Deletion of the herpes simplex virus type 1 ribonucleotide reductase gene alters virulence and latency in vivo . Antiviral Res 17:145–156
    [Google Scholar]
  27. Jacobson J. G., Leib D. A., Goldstein D. J., Bogard C. L., Schaffer P. A., Weller S. K., Coen D. M. 1989; A herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells. Virology 173:276–283
    [Google Scholar]
  28. Johnson R. A., Huong S. M., Huang E. S. 2000; Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38. J Virol 74:1158–1167
    [Google Scholar]
  29. Johnson R. A., Ma X. L., Yurochko A. D., Huang E. S. 2001; The role of MKK1/2 kinase activity in human cytomegalovirus infection. J Gen Virol 82:493–497
    [Google Scholar]
  30. Jordan A., Reichard P. 1998; Ribonucleotide reductases. Annu Rev Biochem 67:71–98
    [Google Scholar]
  31. Kalejta R. F., Shenk T. 2002; Manipulation of the cell cycle by human cytomegalovirus. Front Biosci 7:D295–306
    [Google Scholar]
  32. Kauppi B., Nielsen B. B., Ramaswamy S., Larsen I. K., Thelander M., Thelander L., Eklund H. 1996; The three-dimensional structure of mammalian ribonucleotide reductase protein R2 reveals a more-accessible iron-radical site than Escherichia coli R2. J Mol Biol 262:706–720
    [Google Scholar]
  33. Langelier Y., Champoux L., Hamel M., Guilbault C., Lamarche N., Gaudreau P., Massie B. 1998; The R1 subunit of herpes simplex virus ribonucleotide reductase is a good substrate for host cell protein kinases but is not itself a protein kinase. J Biol Chem 273:1435–1443
    [Google Scholar]
  34. Langelier Y., Bergeron S., Chabaud S., Lippens J., Guilbault C., Sasseville A. M., Denis S., Mosser D. D., Massie B. 2002; The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation. J Gen Virol 83:2779–2789
    [Google Scholar]
  35. Lembo D., Gribaudo G., Hofer A. 7 other authors 2000; Expression of an altered ribonucleotide reductase activity associated with the replication of murine cytomegalovirus in quiescent fibroblasts. J Virol 74:11557–11565
    [Google Scholar]
  36. Lindberg U., Skoog L. 1970; A method for the determination of dATP and dTTP in picomole amounts. Anal Biochem 34:152–160
    [Google Scholar]
  37. Lycksell P. O., Ingemarson R., Davis R., Graslund A., Thelander L. 1994; 1H NMR studies of mouse ribonucleotide reductase: the R2 protein carboxyl-terminal tail, essential for subunit interaction, is highly flexible but becomes rigid in the presence of protein R1. Biochemistry 33:2838–2842
    [Google Scholar]
  38. Maciejewski J. P., Bruening E. E., Donahue R. E., Mocarski E. S., Young N. S., St Jeor S. C. 1992; Infection of hematopoietic progenitor cells by human cytomegalovirus. Blood 80:170–178
    [Google Scholar]
  39. Mocarski E. S. Jr 1996; Cytomegaloviruses and their replication. In Fields Virology , 3rd edn. vol 2 pp  2447–2492 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
    [Google Scholar]
  40. Nordlund P., Eklund H. 1993; Structure and function of the Escherichia coli ribonucleotide reductase protein R2. J Mol Biol 232:123–164
    [Google Scholar]
  41. Paradis H., Gaudreau P., Massie B., Lamarche N., Guilbault C., Gravel S., Langelier Y. 1991; Affinity purification of active subunit 1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity. J Biol Chem 266:9647–9651
    [Google Scholar]
  42. Pari G. S., Anders D. G. 1993; Eleven loci encoding trans -acting factors are required for transient complementation of human cytomegalovirus ori-Lyt-dependent DNA replication. J Virol 67:6979–6988
    [Google Scholar]
  43. Pari G. S., Kacica M. A., Anders D. G. 1993; Open reading frames UL44, IRS1/TRS1, and UL36–38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis. J Virol 67:2575–2582
    [Google Scholar]
  44. Pavloff N., Rivard D., Masson S., Shen S. H., Mes-Masson A. M. 1992; Sequence analysis of the large and small subunits of human ribonucleotide reductase. DNA Seq 2:227–234
    [Google Scholar]
  45. Perkins D., Pereira E. F., Gober M., Yarowsky P. J., Aurelian L. 2002; The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) blocks apoptosis in hippocampal neurons, involving activation of the MEK/MAPK survival pathway. J Virol 76:1435–1449
    [Google Scholar]
  46. Ripalti A., Boccuni M. C., Campanini F., Landini M. P. 1995; Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene. J Virol 69:2047–2057
    [Google Scholar]
  47. Rodems S. M., Spector D. H. 1998; Extracellular signal-regulated kinase activity is sustained early during human cytomegalovirus infection. J Virol 72:9173–9180
    [Google Scholar]
  48. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  49. Sanchez V., Sztul E., Britt W. J. 2000a; Human cytomegalovirus pp28 (UL99) localizes to a cytoplasmic compartment which overlaps the endoplasmic reticulum-golgi-intermediate compartment. J Virol 74:3842–3851
    [Google Scholar]
  50. Sanchez V., Greis K. D., Sztul E., Britt W. J. 2000b; Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly. J Virol 74:975–986
    [Google Scholar]
  51. Schmolke S., Kern H. F., Drescher P., Jahn G., Plachter B. 1995; The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture. J Virol 69:5959–5968
    [Google Scholar]
  52. Sinzger C., Grefte A., Plachter B., Gouw A. S. H., The T. H., Jahn G. 1995; Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. J Gen Virol 76:741–750
    [Google Scholar]
  53. Skaletskaya A., Bartle L. M., Chittenden T., McCormick A. L., Mocarski E. S., Goldmacher V. S. 2001; A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc Natl Acad Sci U S A 98:7829–7834
    [Google Scholar]
  54. Skoog L. 1970; An enzymatic method for the determination of dCTP and dGTP in picomole amounts. Eur J Biochem 17:202–208
    [Google Scholar]
  55. Smith C. C., Aurelian L. 1997; The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is associated with the virion tegument and has PK activity. Virology 234:235–242
    [Google Scholar]
  56. Smith C. C., Peng T., Kulka M., Aurelian L. 1998; The PK domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for immediate-early gene expression and virus growth. J Virol 72:9131–9141
    [Google Scholar]
  57. Smith C. C., Nelson J., Aurelian L., Gober M., Goswami B. B. 2000; Ras-GAP binding and phosphorylation by herpes simplex virus type 2 RR1 PK (ICP10) and activation of the Ras/MEK/MAPK mitogenic pathway are required for timely onset of virus growth. J Virol 74:10417–10429
    [Google Scholar]
  58. Soderberg-Naucler C., Fish K. N., Nelson J. A. 1997; Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 91:119–126
    [Google Scholar]
  59. Somia N. V., Schmitt Miyoshi M. J. H., Verma I. M. 2000; Retroviral vector targeting to human immunodeficiency virus type 1-infected cells by receptor pseudotyping. J Virol 74:4420–4424
    [Google Scholar]
  60. Song Y. J., Stinski M. F. 2002; Effect of the human cytomegalovirus IE86 protein on expression of E2F-responsive genes: a DNA microarray analysis. Proc Natl Acad Sci U S A 99:2836–2841
    [Google Scholar]
  61. Stubbe J., Ge J., Yee C. S. 2001; The evolution of ribonucleotide reduction revisited. Trends Biochem Sci 26:93–99
    [Google Scholar]
  62. Sun Y., Conner J. 1999; The U28 ORF of human herpesvirus-7 does not encode a functional ribonucleotide reductase R1 subunit. J Gen Virol 80:2713–2718
    [Google Scholar]
  63. Tanaka H., Arakawa H., Yamaguchi T., Shiraishi K., Fukuda S., Matsui K., Takei Y., Nakamura Y. 2000; A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 404:42–49
    [Google Scholar]
  64. Uhlin U., Eklund H. 1994; Structure of ribonucleotide reductase protein R1. Nature 370:533–539
    [Google Scholar]
  65. Yamaguchi T., Matsuda K., Sagiya Y., Iwadate M., Fujino M. A., Nakamura Y., Arakawa H. 2001; p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint. Cancer Res 61:8256–8262
    [Google Scholar]
  66. Yu Y., Alwine J. C. 2002; Human cytomegalovirus major immediate-early proteins and simian virus 40 large T antigen can inhibit apoptosis through activation of the phosphatidylinositide 3′-OH kinase pathway and the cellular kinase Akt. J Virol 76:3731–3738
    [Google Scholar]
  67. Zhu H., Shen Y., Shenk T. 1995; Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J Virol 69:7960–7970
    [Google Scholar]
/content/journal/jgv/10.1099/vir.0.19452-0
Loading
/content/journal/jgv/10.1099/vir.0.19452-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

PDF

Supplementary material 3

PDF
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