Department of Virology I1 and Division of Genetic Resources2, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku, Tokyo, 162-8640, Japan
Department of Microbiology, Yamanashi Institute of Health, Fujimi 1-7-31, Kofu City, Yamanashi Prefecture, Japan3
Author for correspondence: Kazuo Yanagi. Fax +81 3 5285 1180. e-mail kyanagi{at}nih.go.jp
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Abstract |
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Main text |
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EBNA-1, which is present at constant levels throughout the cell cycle, is detected by immunofluorescence analyses as diffused and punctate spots in the nuclei of interphase cells. EBNA-1 binds diffusely to mitotic chromosomes (Grogan et al., 1983 ; Marechal et al., 1999
; Petti et al., 1990
). Three separate domains of EBNA-1, aa 854, 7284 and 328365, are required for its binding to metaphase chromosomes (Marechal et al., 1999
). The binding of EBNA-1 to chromosomes is considered to facilitate the partition of a low copy number of latent EBV plasmids, which replicate once per cell cycle, to dividing cells (Marechal et al., 1999
).
In this study, we have investigated whether EBNA-1 colocalizes with cellular chromatin in interphase in vivo using a combined approach of confocal laser-scanning microscopy (LSM) of EBNA-1 fused to green fluorescent protein (GFPEBNA-1), a premature chromosome condensation (PCC) procedure, which facilitates the visualization of chromatin in interphase cells (Gotoh et al., 1995 ; Johnson & Rao, 1970
; Rao, 1977
), and pulse-labelling of cellular DNAs with BrdU or Cy3-dUTP.
The CHO-K1 cell line (originally from the ATCC) was obtained via the Japanese Cancer Research Resources Bank (Tokyo). To construct the GFPEBNA-1 fusion protein, a truncated EBNA-1 lacking the GlyAla copolymer was cleaved from plasmid p205 (Yates et al., 1985 ) with BamHI/HindIII and inserted into the pEGFP-C3 vector (Clontech) at its BglII and HindIII sites (Fig. 1A
).
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Digital images of the fluorescent profiles were acquired on an Axiovert 100M LSM equipped with argon and helium-neon laser light sources using the LSM510 system software, which included the palette option (Zeiss). The peak UV excitation wavelengths were 351 and 364 nm. An objective lens of C-Apochromat 63 x/1·2w corr for optical correction was used for water immersion examination.
For BrdU labelling, cells were incubated in medium containing 10 µM BrdU (Sigma) for 30 min at 37 °C and then in BrdU-free medium for 3 h at 37 °C. These BrdU pulse-labelled cells were treated with calyculin A, swollen under hypotonic conditions, fixed with methanolacetic acid (3:1) and spread onto glass slides. The fluorescence of GFP was observed as described above. The glass slides were treated with 100 units/ml DNase I (Takara) in 100 mM sodium acetate (pH 5·3) and 5 mM magnesium sulfate at 37 °C for 60 min, according to Carayon & Bord (1992) . The PCCs were then incubated with an anti-BrdU mouse monoclonal antibody (MBL) followed by Texas red-conjugated donkey anti-mouse IgG antibodies (Jackson Laboratories).
Cy3-dUTP (Pharmacia) was incorporated into monolayer cells according to the methods of McNeil & Warder (1987) and Manders et al. (1999)
. Briefly, Cy3-dUTP was added at 10 µM to cells and 425600 µm diameter glass beads (Sigma) were immediately sprinkled onto the cells. The cell culture covered with the beads was tapped and the beads were washed out immediately. The cells were then incubated in fresh medium.
The GFPEBNA-1 fusion protein colocalizes with cellular chromatin that is prematurely condensed during interphase. The GFPEBNA-1 fusion protein lacking the GlyAla copolymer, which is not required for the replication and maintenance of EBV DNA (Yates et al., 1985 ) or for metaphase chromosome binding (Marechal et al., 1999
), was expressed in CHO-K1 cells; chromosomes of CHO-K1 cells are more clearly visible owing to their small number (20 per cell) and correspondingly large size. The 84 kDa GFPEBNA-1 protein was detected in extracts from GFPEBNA-1-expressing CHO-K1 cells by Western blotting (Fig. 1B
). GFPEBNA-1 was localized in intranuclear granules or spots, similar to those of full-length B95-8 EBNA-1 expressed in CHO-K1 cells (Fig. 1C
). LSM confocal images of GFPEBNA-1 and DNA showed that GFPEBNA-1 colocalized with mitotic chromosomes in living cells, which is in agreement with the results of Marechal et al. (1999)
(Fig. 1C
, a
).
To visualize chromatin in interphase cells, we took advantage of the PCC procedure, which was discovered following the cell fusion of interphase cells with mitotic ones (Johnson & Rao, 1970 ; Rao, 1977
). Recently, chemical procedures have been developed to induce PCC using the phosphatase inhibitors calyculin A, fostriecin and okadaic acid (Coco-Martin & Begg, 1997
; Gotoh et al., 1995
; Guo et al., 1995
). These reagents induce PCC at a given stage of cell division, bringing about various forms of PCCs that are characteristic of each phase or stage of the cell cycle (Alsbeih & Raaphorst, 1999
; Coco-Martin & Begg, 1997
; Gotoh et al., 1995
).
We analysed the distribution pattern of EBNA-1 throughout the process of calyculin A-induced PCC in living GFPEBNA-1-expressing CHO-K1 cells by maintaining a dish of cultured cells under the microscope. The GFPEBNA-1 protein was concentrated together with chromosomal DNAs onto PCCs throughout the PCC process, as shown in the early (Fig. 2A, b
) and final (Fig. 2A
, c
) stages of PCC [note that cells shown in Fig. 2(A
, a
c
) are different cells from the same dish]. In contrast, control GFP remained diffuse after treatment with calyculin A (Fig. 2A
, e
and f
).
|
|
Next, we pulse-labelled cellular DNAs with Cy3-dUTP to examine the localization of GFPEBNA-1 on PCCs in living cells. The Cy3-labelled patterns were similar to those reported by Sadoni et al. (1999) . Confocal LSM of Cy3-labelled GFPEBNA-1-expressing CHO-K1 cells, which were maintained in culture medium throughout the microscopic observations, demonstrated that EBNA-1 colocalized well with the Cy3-labelled regions of cellular chromatin in living cells (Fig. 3B
, a
and b
, top rows); the same cells were treated with calyculin A, observed continually under the microscope and sequential photographs of the PCCs of the same cells were taken. The photographs taken at the indicated time after the addition of calyculin A indicated that GFPEBNA-1 remained associated with the Cy3-labelled, recently replicated regions of chromatin throughout the PCC process (Fig. 3B
).
The binding of EBNA-1 to metaphase chromosomes is considered to be involved in the segregation of a low copy number of EBV plasmids in dividing cells (Hung et al., 2001 ; Marechal et al., 1999
). EBNA-1 of herpesvirus papio and LANA of Kaposi's sarcoma-associated herpesvirus, proteins that are necessary for the persistence of these viral episomes, also bind to mitotic chromosomes (Ballestas et al., 1999
; Piolot et al., 2001
).
In the studies presented here, we have shown that EBNA-1 colocalizes with cellular chromatin that is condensed during interphase in the absence of oriP DNA by using a combined approach of confocal microscopy of a GFPEBNA-1 fusion protein, PCC and pulse-labelling of cellular replicating DNAs. The results of our studies in living cells expand previous biochemical data showing that EBNA-1 is found in chromatin fractions from both EBV latently infected cells (Petti et al., 1990 ) and cells containing no oriP plasmids (Kanda et al., 2001
) because isolation or purification of intact chromatin is difficult (reviewed by Cook, 2001
). Moreover, we have shown that GFPEBNA-1 is highly colocalized with recently replicated regions of cellular chromatin in S phase in particular. EBNA-1 might bind to a cellular chromatin/chromosome protein during normal condensation in the mitotic prophase but it seems less likely because our observations in this study showed that GFPEBNA-1 highly colocalized with not only the PCCs that were formed during G2 phase but also those formed during G1 and S phases. Another possibility that GFPEBNA-1 bound to PCCs only after the induction of premature condensation is also less likely because GFPEBNA-1 was as highly colocalized with Cy3-labelled regions of cellular DNA before the induction of PCC as during and after PCC (Fig. 3B
). Thus, we conclude that EBNA-1 is likely to be associated with interphase chromatin in living cells and, in particular, with its newly replicated regions. Further studies on the interaction of EBNA-1 with chromatin/chromosomes and their components during the course of the cell division cycle should provide better understanding of the molecular mechanism of the multiple functions of this key protein in EBV latency.
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Acknowledgments |
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References |
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Alsbeih, G. & Raaphorst, G. P. (1999). Differential induction of premature chromosome condensation by calyculin A in human fibroblast and tumor cell lines. Anticancer Research 19, 903-908.[Medline]
Ballestas, M. E., Chatis, P. A. & Kaye, K. M. (1999). Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284, 641-644.
Carayon, P. & Bord, A. (1992). Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA. Journal of Immunological Methods 147, 225-230.[Medline]
Coco-Martin, J. M. & Begg, A. C. (1997). Detection of radiation-induced chromosome aberrations using fluorescence in situ hybridization in drug-induced premature chromosome condensations of tumour cell lines with different radiosensitivities. International Journal of Radiation Biology 71, 265-273.[Medline]
Cook, P. R. (2001). Principles of Nuclear Structure and Function. New York: Wiley-Liss.
Fujita, T., Ikeda, M., Kusano, S., Yamazaki, M., Ito, S., Obayashi, M. & Yanagi, K. (2001). Amino acid substitution analyses of the DNA contact region, two amphipathic alpha-helices and a recognition-helix-like helix outside the dimeric beta-barrel of EpsteinBarr virus nuclear antigen 1. Intervirology 44, 271-282.[Medline]
Gotoh, E., Asakawa, Y. & Kosaka, H. (1995). Inhibition of protein serine/threonine phosphatases directly induces premature chromosome condensation in mammalian somatic cells. Biomedical Research 16, 63-68.
Grogan, E. A., Summers, W. P., Dowling, S., Shedd, D., Gradoville, L. & Miller, G. (1983). Two EpsteinBarr viral nuclear neoantigens distinguished by gene transfer, serology, and chromosome binding. Proceedings of the National Academy of Sciences, USA 80, 7650-7653.[Abstract]
Guo, X. W., Th'ng, J. P., Swank, R. A., Anderson, H. J., Tudan, C., Bradbury, E. M. & Roberge, M. (1995). Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation. EMBO Journal 14, 976-985.[Abstract]
Hung, S. C., Kang, M. S. & Kieff, E. (2001). Maintenance of EpsteinBarr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1. Proceedings of the National Academy of Sciences, USA 98, 1865-1870.
Ito, S., Ikeda, M., Kato, N., Matsumoto, A., Ishikawa, Y., Kumakubo, S. & Yanagi, K. (2000). EpsteinBarr virus nuclear antigen-1 binds to nuclear transporter karyopherin alpha1/NPI-1 in addition to karyopherin alpha2/Rch1. Virology 266, 110-119.[Medline]
Johnson, R. T. & Rao, P. N. (1970). Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature 226, 717-722.[Medline]
Kanda, T., Otter, M. & Wahl, G. M. (2001). Coupling of mitotic chromosome tethering and replication competence in EpsteinBarr virus-based plasmids. Molecular and Cellular Biology 21, 3576-3588.
Kieff, E. & Rickinson, A. B. (2001). EpsteinBarr virus and its replication. In Fields Virology , pp. 2511-2573. Edited by D. M. Knipe & P. M. Howley. Philadelphia:Lippincott Williams & Wilkins.
Kube, D., Vockerodt, M., Weber, O., Hell, K., Wolf, J., Haier, B., Grasser, F. A., Muller-Lantzsch, N., Kieff, E., Diehl, V. & Tesch, H. (1999). Expression of EpsteinBarr virus nuclear antigen 1 is associated with enhanced expression of CD25 in the Hodgkin cell line L428. Journal of Virology 73, 1630-1636.
Kusano, S., Tamada, K., Senpuku, H., Harada, S., Ito, S. & Yanagi, K. (2001). EpsteinBarr virus nuclear antigen-1-dependent and -independent oriP-binding cellular proteins. Intervirology 44, 283-290.[Medline]
Mackey, D. & Sugden, B. (1999). The linking regions of EBNA1 are essential for its support of replication and transcription. Molecular and Cellular Biology 19, 3349-3359.
McNeil, P. L. & Warder, E. (1987). Glass beads load macromolecules into living cells. Journal of Cell Science 88, 669-678.[Abstract]
Manders, E. M., Kimura, H. & Cook, P. R. (1999). Direct imaging of DNA in living cells reveals the dynamics of chromosome formation. Journal of Cell Biology 144, 813-821.
Marechal, V., Dehee, A., Chikhi-Brachet, R., Piolot, T., Coppey-Moisan, M. & Nicolas, J. C. (1999). Mapping EBNA-1 domains involved in binding to metaphase chromosomes. Journal of Virology 73, 4385-4392.
Nonkwelo, C., Skinner, J., Bell, A., Rickinson, A. & Sample, J. (1996). Transcription start sites downstream of the EpsteinBarr virus (EBV) Fp promoter in early-passage Burkitt lymphoma cells define a fourth promoter for expression of the EBV EBNA-1 protein. Journal of Virology 70, 623-627.[Abstract]
Petti, L., Sample, C. & Kieff, E. (1990). Subnuclear localization and phosphorylation of EpsteinBarr virus latent infection nuclear proteins. Virology 176, 563-574.[Medline]
Piolot, T., Tramier, M., Coppey, M., Nicolas, J. C. & Marechal, V. (2001). Close but distinct regions of human herpesvirus 8 latency-associated nuclear antigen 1 are responsible for nuclear targeting and binding to human mitotic chromosomes. Journal of Virology 75, 3948-3959.
Rao, P. N. (1977). Premature chromosome condensation and the fine structure of chromosomes. In Molecular Structure of Human Chromosomes , pp. 205-231. Edited by J. J. Yunis. New York:Academic Press.
Rickinson, A. B. & Kieff, E. (2001). EpsteinBarr Virus. In Fields Virology , pp. 2575-2627. Edited by D. M. Knipe & P. M. Howley. Philadelphia:Lippincott Williams & Wilkins.
Sadoni, N., Langer, S., Fauth, C., Bernardi, G., Cremer, T., Turner, B. M. & Zink, D. (1999). Nuclear organization of mammalian genomes. Polar chromosome territories build up functionally distinct higher order compartments. Journal of Cell Biology 146, 1211-1226.
Shire, K., Ceccarelli, D. F., Avolio-Hunter, T. M. & Frappier, L. (1999). EBP2, a human protein that interacts with sequences of the EpsteinBarr virus nuclear antigen 1 important for plasmid maintenance. Journal of Virology 73, 2587-2595.
Sugden, B. & Warren, N. (1989). A promoter of EpsteinBarr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection. Journal of Virology 63, 2644-2649.[Medline]
Ward, W. W. & Bokman, S. H. (1982). Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21, 4535-4540.[Medline]
Yates, J. L. (1996). EpsteinBarr virus DNA replication. In DNA Replication in Eukaryotic Cells , pp. 751-774. Edited by M. L. DePamphilis. New York:Cold Spring Harbor Laboratory.
Yates, J. L., Warren, N. & Sugden, B. (1985). Stable replication of plasmids derived from EpsteinBarr virus in various mammalian cells. Nature 313, 812-815.[Medline]
Yates, J. L., Camiolo, S. M. & Bashaw, J. M. (2000). The minimal replicator of EpsteinBarr virus oriP. Journal of Virology 74, 4512-4522.
Zhang, D., Frappier, L., Gibbs, E., Hurwitz, J. & ODonnell, M. (1998). Human RPA (hSSB) interacts with EBNA1, the latent origin binding protein of EpsteinBarr virus. Nucleic Acids Research 26, 631-637.
Received 5 March 2002;
accepted 6 May 2002.