1887

Abstract

Human cytomegalovirus (HCMV) DNA-binding protein pUL56 is thought to be involved in the cleavage/packaging process of viral DNA and therefore needs to be transported into the nucleus. By using indirect immunofluorescence analysis, HCMV pUL56 (p130) was found to be localized predominantly in the nucleus of infected cells. Solitary expression of wild-type as well as epitope-tagged pUL56 also resulted in nuclear distribution after transfection, suggesting the presence of an endogenous nuclear localization signal (NLS). Deletion of a carboxy-terminal stretch of basic amino acids (aa 816–827) prevented nuclear translocation, indicating that the sequence RRVRATRKRPRR of HCMV pUL56 mediates nuclear targetting. The signal character of the NLS sequence was demonstrated by successful transfer of the NLS to a reporter protein chimera. Furthermore, sequential substitutions of pairs of amino acids by alanine in the context of the reporter protein as well as substitutions within the full-length pUL56 sequence indicated that residues at positions 7 and 8 of the NLS (R and K at positions 822 and 823 of pUL56) were essential for nuclear translocation. In order to identify the transport machinery involved, the potential of pUL56 to bind importin α (hSRP1α) was examined. Clear evidence of a direct interaction of a carboxy-terminal portion as well as the NLS of pUL56 with hSRP1α was provided by binding assays. In view of these findings, it is suggested that nuclear translocation of HCMV pUL56 is mediated by the importin-dependent pathway.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-81-9-2231
2000-09-01
2019-10-18
Loading full text...

Full text loading...

/deliver/fulltext/jgv/81/9/0812231a.html?itemId=/content/journal/jgv/10.1099/0022-1317-81-9-2231&mimeType=html&fmt=ahah

References

  1. Adam, S. A. & Gerace, L. ( 1991; ). Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell 66, 837-847.[CrossRef]
    [Google Scholar]
  2. Adam, S. A., Lobl, T. J., Mitchell, M. A. & Gerace, L. ( 1989; ). Identification of specific binding proteins for a nuclear location sequence. Nature 337, 276-279.[CrossRef]
    [Google Scholar]
  3. Addison, C., Rixon, F. J. & Preston, V. G. ( 1990; ). Herpes simplex virus type 1 UL28 gene product is important for the formation of mature capsids. Journal of General Virology 71, 2377-2384.[CrossRef]
    [Google Scholar]
  4. Ambinder, R. F., Mullen, M. A., Chang, Y. N., Hayward, G. S. & Hayward, S. D. ( 1991; ). Functional domains of Epstein–Barr virus nuclear antigen EBNA-1. Journal of Virology 65, 1466-1478.
    [Google Scholar]
  5. Blanton, R. A. & Tevethia, M. J. ( 1981; ). Immunoprecipitation of virus-specific immediate-early and early polypeptides from cells lytically infected with human cytomegalovirus strain AD 169. Virology 112, 262-273.[CrossRef]
    [Google Scholar]
  6. Bogner, E. ( 1999; ). Human cytomegalovirus nuclease: implications for new strategies in gene therapy. Genetics and Molecular Biology 3, 75-78.
    [Google Scholar]
  7. Bogner, E., Reschke, M., Reis, B., Reis, E., Britt, W. & Radsak, K. ( 1992; ). Recognition of compartmentalized intracellular analogs of glycoprotein H of human cytomegalovirus. Archives of Virology 126, 67-80.[CrossRef]
    [Google Scholar]
  8. Bogner, E., Reschke, M., Reis, B., Mockenhaupt, T. & Radsak, K. ( 1993; ). Identification of the gene product encoded by ORF UL56 of the human cytomegalovirus genome. Virology 196, 290-293.[CrossRef]
    [Google Scholar]
  9. Bogner, E., Radsak, K. & Stinski, M. F. ( 1998; ). The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity. Journal of Virology 72, 2259-2264.
    [Google Scholar]
  10. Bonner, W. M. & Laskey, R. A. ( 1974; ). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. European Journal of Biochemistry 46, 83-88.[CrossRef]
    [Google Scholar]
  11. Boulikas, T. ( 1993; ). Nuclear localization signals (NLS). Critical Reviews in Eukaryotic Gene Expression 3, 193-227.
    [Google Scholar]
  12. Britt, W. J. & Alford, C. A. ( 1996; ). Cytomegalovirus. In Fields Virology, pp. 2493-2523. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.
  13. Chang, D., Haynes, J. I.II, Brady, J. N. & Consigli, R. A. ( 1992; ). The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1. Virology 189, 821-827.[CrossRef]
    [Google Scholar]
  14. Chee, M. S., Bankier, A. T., Beck, S., Bohni, R., Brown, C. M., Cerny, R., Horsnell, T., Hutchison, C. A.III, Kouzarides, T., Martignetti, J. A., Preddie, E., Sathwell, S. C., Tomlinson, P., Weston, K. M. & Barell, B. G. ( 1990; ). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Current Topics in Microbiology and Immunology 154, 125-169.
    [Google Scholar]
  15. Chelsky, D., Ralph, R. & Jonak, G. ( 1989; ). Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Molecular and Cellular Biology 9, 2487-2492.
    [Google Scholar]
  16. 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]
  17. Dang, C. V. & Lee, W. M. ( 1989; ). Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins. Journal of Biological Chemistry 264, 18019-18023.
    [Google Scholar]
  18. Dingwall, C. & Laskey, R. A. ( 1991; ). Nuclear targeting sequences – a consensus? Trends in Biochemical Sciences 16, 478-481.[CrossRef]
    [Google Scholar]
  19. Fischer, N., Kremmer, E., Lautscham, G., Mueller-Lantzsch, N. & Grasser, F. A. ( 1997; ). Epstein–Barr virus nuclear antigen 1 forms a complex with the nuclear transporter karyopherin α2. Journal of Biological Chemistry 272, 3999-4005.[CrossRef]
    [Google Scholar]
  20. Gibson, W. ( 1996; ). Structure and assembly of the virion. Intervirology 39, 389-400.
    [Google Scholar]
  21. Giesen, K., Radsak, K. & Bogner, E. ( 2000; ). Targeting of the gene product encoded by ORF UL56 of human cytomegalovirus into viral replication centers. FEBS Letters 471, 215-218.[CrossRef]
    [Google Scholar]
  22. Görlich, D. ( 1997; ). Nuclear protein import. Current Opinion in Cell Biology 9, 412-419.[CrossRef]
    [Google Scholar]
  23. Görlich, D., Kostka, S., Kraft, R., Dingwall, C., Laskey, R. A., Hartmann, E. & Prehn, S. ( 1995; ). Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Current Biology 5, 383-392.
    [Google Scholar]
  24. Görlich, D., Panté, N., Kutay, U., Aebi, U. & Bischoff, F. R. ( 1996; ). Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO Journal 15, 5584-5594.
    [Google Scholar]
  25. Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. ( 1984; ). A short amino acid sequence able to specify nuclear location. Cell 39, 499-509.[CrossRef]
    [Google Scholar]
  26. Köhler, M., Speck, C., Christiansen, M., Bischoff, F. R., Prehn, S., Haller, H., Görlich, D. & Hartmann, E. ( 1999; ). Evidence for distinct substrate specificities of importin α family members in nuclear protein import. Molecular and Cellular Biology 19, 7782-7791.
    [Google Scholar]
  27. Lobl, T. J., Mitchell, M. A. & Maggiora, L. L. ( 1990; ). SV40 large T-antigen nuclear signal analogues: successful nuclear targeting with bovine serum albumin but not low molecular weight fluorescent conjugates. Biopolymers 29, 197-203.[CrossRef]
    [Google Scholar]
  28. Makkerh, J. P. S., Dingwall, C. & Laskey, R. A. ( 1996; ). Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids. Current Biology 6, 1025-1027.[CrossRef]
    [Google Scholar]
  29. Mears, W. E., Lam, V. & Rice, S. A. ( 1995; ). Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27. Journal of Virology 69, 935-947.
    [Google Scholar]
  30. Melchior, F., Paschal, B., Evans, J. & Gerace, L. ( 1993; ). Inhibition of nuclear protein import by nonhydrolyzable analogs of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. Journal of Cell Biology 123, 1649-1659.[CrossRef]
    [Google Scholar]
  31. Mettenleiter, T. C., Saalmüller, A. & Weiland, F. ( 1993; ). Pseudorabies virus protein homologous to herpes simplex virus type 1 ICP 18.5 is necessary for capsid maturation. Journal of Virology 67, 1236-1245.
    [Google Scholar]
  32. Mocarski, E. S.Jr ( 1996; ). Cytomegaloviruses and their replication. In Fields Virology, pp. 2447-2492. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.
  33. Moore, M. S. & Blobel, G. ( 1994; ). Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proceedings of the National Academy of Sciences, USA 91, 10212-10216.[CrossRef]
    [Google Scholar]
  34. Moroianu, J., Hijikata, M., Blobel, G. & Radu, A. ( 1995; ). Mammalian karyopherin α1β and α2β heterodimers: α1 or α2 subunit binds nuclear localization sequence and β subunit interacts with peptide repeat-containing nucleoporins. Proceedings of the National Academy of Sciences, USA 92, 6532-6536.[CrossRef]
    [Google Scholar]
  35. Newmeyer, D. D., Lucocq, J. M., Bürglin, T. R. & De Robertis, E. M. ( 1986; ). Assembly in vitro of nuclei active in nuclear transport: ATP is required for nucleoplasmin accumulation. EMBO Journal 5, 501-510.
    [Google Scholar]
  36. Nosaka, T., Siomi, H., Adachi, Y., Ishibashi, M., Kubota, S., Maki, M. & Hatanaka, M. ( 1989; ). Nucleolar targeting signal of human T-cell leukaemia virus type I rex-encoded protein is essential for cytoplasmic accumulation of unspliced viral mRNA. Proceedings of the National Academy of Sciences, USA 86, 9798-9802.[CrossRef]
    [Google Scholar]
  37. Pederson, N. E. & Enquist, L. W. ( 1989; ). The nucleotide sequence of a pseudorabies virus gene similar to ICP18.5 of herpes simplex virus type 1. Nucleic Acids Research 17, 3597.[CrossRef]
    [Google Scholar]
  38. Pederson, N. E. & Enquist, L. W. ( 1991; ). Overexpression in bacteria and identification in infected cells of the pseudorabies virus protein homologous to herpes simplex virus type 1 ICP18.5. Journal of Virology 65, 3746-3758.
    [Google Scholar]
  39. Pellett, P. E., Jenkins, F. J., Ackermann, M., Sarmiento, M. & Roizman, B. ( 1986; ). Transcription initiation sites and nucleotide sequence of a herpes simplex virus 1 gene conserved in the Epstein–Barr virus genome and reported to affect the transport of viral glycoproteins. Journal of Virology 60, 1134-1140.
    [Google Scholar]
  40. Penfold, M. E. & Mocarski, E. S. ( 1997; ). Formation of cytomegalovirus DNA replication compartments defined by localization of viral proteins and DNA synthesis. Virology 239, 46-61.[CrossRef]
    [Google Scholar]
  41. Plafker, S. M. & Gibson, W. ( 1998; ). Cytomegalovirus assembly protein precursor and proteinase precursor contain two nuclear localization signals that mediate their own nuclear translocation and that of the major capsid protein. Journal of Virology 72, 7722-7732.
    [Google Scholar]
  42. Radsak, K., Brücher, K. H., Britt, W., Shiou, H., Schneider, D. & Kollert, A. ( 1990; ). Nuclear compartmentation of glycoprotein B of human cytomegalovirus. Virology 177, 515-522.[CrossRef]
    [Google Scholar]
  43. Radsak, K., Eickmann, M., Mockenhaupt, T., Bogner, E., Kern, H., Eis-Hübinger, A. & Reschke, M. ( 1996; ). Retrieval of human cytomegalovirus glycoprotein B from the infected cell surface for virus envelopment. Archives of Virology 141, 557-572.[CrossRef]
    [Google Scholar]
  44. Richardson, W. D., Roberts, B. L. & Smith, A. E. ( 1986; ). Nuclear location signals in polyoma virus large-T. Cell 44, 77-85.[CrossRef]
    [Google Scholar]
  45. Robbins, J., Dilworth, S. M., Laskey, R. A. & Dingwall, C. ( 1991; ). Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64, 615-623.[CrossRef]
    [Google Scholar]
  46. Roberts, B. L., Richardson, W. D. & Smith, A. E. ( 1987; ). The effect of protein context on nuclear location signal function. Cell 50, 465-475.[CrossRef]
    [Google Scholar]
  47. Rost, B. & Sander, C. ( 1993; ). Prediction of protein secondary structure at better than 70% accuracy. Journal of Molecular Biology 232, 584-599.[CrossRef]
    [Google Scholar]
  48. Sarisky, R. T. & Hayward, G. S. ( 1996; ). Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLyt-dependent DNA replication and formation of replication compartments in cotransfection assays. Journal of Virology 70, 7398-7413.
    [Google Scholar]
  49. Schmidt-Zachmann, M. S. & Nigg, E. A. ( 1993; ). Protein localization to the nucleolus: a search for targeting domains in nucleolin. Journal of Cell Science 105, 799-806.
    [Google Scholar]
  50. Schmolke, S., Drescher, P., Jahn, G. & Plachter, B. ( 1995; ). 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. Journal of Virology 69, 1071-1078.
    [Google Scholar]
  51. Siomi, H., Shida, H., Nam, S. H., Nosaka, T., Maki, M. & Hatanaka, M. ( 1988; ). Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing. Cell 55, 197-209.[CrossRef]
    [Google Scholar]
  52. Smuda, Ch., Bogner, E. & Radsak, K. ( 1997; ). The human cytomegalovirus glycoprotein B gene (ORF UL55) is expressed early in the infectious cycle. Journal of General Virology 78, 1981-1992.
    [Google Scholar]
  53. Sorg, G. & Stamminger, T. ( 1999; ). Mapping of nuclear localization signals by simultaneous fusion to green fluorescent protein and to beta-galactosidase. Biotechniques 26, 858-862.
    [Google Scholar]
  54. Tengelsen, L. A., Pederson, N. E., Shaver, P. R., Wathen, M. W. & Homa, F. L. ( 1993; ). Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants. Journal of Virology 67, 3470-3480.
    [Google Scholar]
  55. Warner, A. K. & Sloboda, R. D. ( 1999; ). C-terminal domain of the mitotic apparatus protein p62 targets the protein to the nucleolus during interphase. Cell Motility and the Cytoskeleton 44, 68-80.[CrossRef]
    [Google Scholar]
  56. Weis, K., Mattaj, I. W. & Lamond, A. I. ( 1995; ). Identification of hSRP1α as a functional receptor for nuclear localization sequences. Science 268, 1049-1053.[CrossRef]
    [Google Scholar]
  57. Weis, K., Ryder, U. & Lamond, A. I. ( 1996a; ). The conserved amino-terminal domain of hSRP1α is essential for nuclear protein import. EMBO Journal 15, 1818-1825.
    [Google Scholar]
  58. Weis, K., Dingwall, C. & Lamond, A. I. ( 1996b; ). Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities. EMBO Journal 15, 7120-7128.
    [Google Scholar]
  59. Zasloff, M. ( 1983; ). tRNA transport from the nucleus in a eukaryotic cell: carrier-mediated translocation process. Proceedings of the National Academy of Sciences, USA 80, 6436-6440.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-81-9-2231
Loading
/content/journal/jgv/10.1099/0022-1317-81-9-2231
Loading

Data & Media loading...

Most Cited This Month

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