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Journal of Virology, February 2006, p. 2045-2050, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.2045-2050.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

cdc2/Cyclin B1-Dependent Phosphorylation of EBNA2 at Ser243 Regulates Its Function in Mitosis

Wei Yue,1 Julia Shackelford,1 and Joseph S. Pagano1,2,3*

Department of Medicine,2 Department of Microbiology and Immunology,3 Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275991

Received 2 August 2005/ Accepted 18 November 2005


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ABSTRACT
 
Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) transactivates EBV genes in latently infected B cells. We have shown that mitotic hyperphosphorylation of EBNA2 suppresses its ability to transactivate the latent membrane protein 1 (LMP1) promoter. In this follow-up study, we identify EBNA2 Ser243 as a phosphorylation site for mitotic cdc2/cyclin B1 kinase. Mutation at Ser243, which mimics constitutive phosphorylation of the protein, decreases endogenous levels of both LMP1 and EBNA2. Moreover, mutation at Ser243 reduces the ability of EBNA2 to transactivate Cp, the promoter for all six EBV EBNA genes. Our data implicate EBNA2 Ser243 as a cdc2/cyclin B1 site of phosphorylation important for EBNA2's cotranscriptional function in mitosis.


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TEXT
 
Epstein-Barr virus (EBV) is associated with the development of certain malignancies (5, 15). EBV's oncogenic potential is mirrored in vitro, where establishment of latent infection in human B lymphocytes leads to the outgrowth of immortalized lymphoblastoid cell lines (LCLs) (25, 33). EBV gene expression in LCLs is restricted to six nuclear antigens (EBNA1, -2, -3A, -3B, -3C, and -LP), three integral membrane proteins (LMP1, -2A, and -2B) and two nonpolyadenylated RNAs (EBER1 and -2) (reviewed in reference 36). EBNA2 is essential for EBV transformation; EBV with a region of the genome that contains EBNA2 deleted is unable to transform B lymphocytes, and correction of the deletion through genomic recombination restores transforming ability (8). EBNA2 transactivates all of the other latency gene promoters, including Cp, from which all six EBNAs are transcribed (17, 40), and the LMP1 (1, 43) and LMP2A (48, 49) promoters, from which the three latent membrane proteins (LMPs) are transcribed.

EBV maintains life-long latency in host cells. In latently infected LCLs or tumor cells EBV is usually maintained as an episome with copy numbers essentially constant in each cell line (26, 36), and the latent EBV episomes are located in chromosomes (37, 38). There have been extensive reports about transactivation and regulation of EBV latent gene promoters (16, 17, 20, 43); however, most studies were carried out in asynchronously growing cells, and little is known about transcription from EBV episomes in mitosis when transcription from host chromosomes is generally suppressed (13, 34, 41).

We recently reported that in type III latency EBNA2 is hyperphosphorylated specifically in mitosis, which suppresses transactivation of the LMP1 promoter. We implicated cdc2/cyclin B1 kinase in this hyperphosphorylation (46). In this follow-up report, we confirm the importance of hyperphosphorylation of EBNA2 in suppressing its function by identifying Ser243 as a cdc2/cyclin B1 phosphorylation site. Hyperphosphorylation at this site impairs not only transcription of the LMP1 promoter, but also the BamHI C promoter (Cp) used for transcription of all of the EBNAs.

Ser243 on EBNA2 is a phosphorylation site of cdc2/cyclin B1 kinase. The consensus motif for cdc2 kinase is Ser/Thr-Pro-X-Z (where X is a polar amino acid and Z is generally a basic amino acid) (29). In the EBNA2 protein, Ser243 is the most likely candidate site (sequence SPPR). First, we tested whether cdc2/cyclin B1 can directly phosphorylate purified EBNA2 at Ser243 in vitro. Purification of an EBNA2 fragment encoding 185 to 315 amino acids (185-315aa) or 185-315aa S243A, in which Ser243 was substituted with alanine, and in vitro kinase assay were described previously (46, 47). As shown in Fig. 1A, GST-EBNA2 185-315aa fusion protein which contains the potential phosphorylation site Ser243 was phosphorylated by cdc2/cyclin B1, whereas neither Ser243A substitution in the GST-EBNA2 185-315aa nor glutathione S-transferase (GST) was phosphorylated. The results indicate that cdc2/cyclin B1 kinase directly phosphorylates EBNA2 at Ser243 in vitro. Second, we studied whether cdc2/cyclin B1 kinase phosphorylates Ser243 when EBNA2 is expressed in mammalian cells. Wild-type full-length EBNA2 or EBNA2 with Ser243A substitution were immunoprecipitated from HeLa cells transiently transfected with either pSG5 EBNA2 (a gift from P. D. Ling) or pSG5 EBNA2 Ser243A plasmid (47) and subjected to cdc2/cyclin B1 kinase assay. As shown in Fig. 1B, wild-type EBNA2 can be phosphorylated efficiently in the presence of cdc2/cyclin B1 kinase, whereas phosphorylation of EBNA2 Ser243A is greatly diminished. Immunoblot analysis shows that essentially equal amounts of EBNA2 were used in the kinase assays. Taken together, the results shown in Fig. 1A and B indicate that Ser243 of EBNA2 is a specific phosphorylation site for cdc2/cyclin B1 kinase.


Figure 1
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  		FIG. 1. Phosphorylation of cdc2 site Ser243 in vitro and in vivo in mitosis. (A) Phosphorylation of purified GST-EBNA2 185-313aa (GST-E2), GST-EBNA2 185-313aa Ser243A (GST-E2 S243A) or GST in the presence (+) or absence (–) of cdc2/cyclin B1 kinase was carried out. The reaction mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4 to 20% gradient gels). After Coomassie bright blue (CBB) staining, gels were exposed on a PhosphorImager. (B) Transiently expressed wild-type EBNA2 (wt) or EBNA2 S243A in HeLa cells were immunoprecipitated by EBNA2 antibody and used as substrates for cdc2/cyclin B1 kinase assays. Immunocomplexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto membrane; phosphorylation of EBNA2 was detected by autoradiography (top panel). Equal protein levels of EBNA2 were confirmed by immunoblotting (bottom panel). (C) Migration of wild-type EBNA2 and EBNA2 Ser243 in different phases of the cell cycle. HeLa cells were synchronized at the G1/S border by double-thymidine block. S- and G1-phase cells were collected 6 and 24 h after release from thymidine, and M-phase cells were collected by vigorous shaking-off at 10 h after release.

Ser243 is a mitosis-specific site of EBNA2 phosphorylation. Since EBNA2 is hyperphosphorylated specifically in mitosis and cdc2/cyclin B1 is the key regulator of mitosis and has the highest kinase activity in mitosis, we next ascertained whether Ser243 is a mitotic-phosphorylation site of EBNA2 in vivo. Wild-type EBNA2 or EBNA2 Ser243A were transiently expressed in HeLa cells. At 24 h posttransfection, cells were synchronized at the G1/S border by double-thymidine blockade (6). Mitotic cells were collected by vigorous shaking-off 10 h after release from thymidine block. S-phase and G1-phase cells (as determined by flow cytometry, data not shown) were collected at 6 and 24 h postrelease, respectively. As exemplified in Fig. 1C, migration of EBNA2 Ser243A was consistently faster than that of wild-type EBNA2 in mitotic cells obtained by the shake-off method. Similar results were also obtained in nocodazole-arrested mitotic cells (data not shown); however, no obvious difference in mobility of these two proteins was detected in interphase cells. {lambda}-Phosphatase assays performed as described previously (46) confirmed that the shift in migration of EBNA2 and EBNA2 S243A in mitosis was due to differences in phosphorylation (data not shown). Therefore, the results indicate that Ser243 is a mitosis-specific hyperphosphorylation site of EBNA2 in vivo.

Phosphorylation at Ser243 suppresses EBNA2 transactivation function. First, we studied whether phosphorylation of EBNA2 at Ser243 suppresses its ability to induce expression of LMP1. P3HR1 is an EBV-positive cell line in which EBNA-LP and EBNA2 open reading frames are deleted (9), and it expresses only low levels of LMP1; transfected EBNA2 can transactivate the endogenous LMP1 promoter and induce LMP1 expression in this cell line (30). Since replacement of serine by aspartic acid (D) (24, 35) or glutamic acid (E) (22, 23) can mimic phosphorylation, Ser243 of EBNA2 was mutated to aspartic acid (D) or glutamic acid (E), and the ability of these mutated proteins to induce LMP1 expression was compared when they were exogenously expressed in P3HR1 cells. As shown in Fig. 2A, wild-type EBNA2 efficiently induces LMP1 protein expression compared to empty vector; however, induction of LMP1 by the EBNA2 Ser243D or Ser243E mutant proteins is dramatically suppressed.


Figure 2
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  		FIG. 2. Induction of endogenous LMP1 expression in P3HR1 cells by EBNA2. Wild-type EBNA2, EBNA2 Ser243D, or EBNA2 Ser243E were transiently expressed in P3HR1 cells through electroporation. Transfection with empty vector was used as control. (A) Whole-cell lysates were subjected to immunoblotting for LMP1 and EBNA2 48 h after transfection. ß-Actin was used as a loading control. (B) Transfected cells were selected with anti-CD4 magnetic beads, and total RNA was extracted and subjected to RPA for LMP1 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) RNAs. Yeast RNA was used as negative and X50-7 RNA positive controls. A histogram quantifying LMP1 mRNA signals by normalization with GAPDH mRNA signals is shown. (C) Immunoblotting of EBNA2 in P3HR1 cells after nuclear and cytoplasm separation 48 h posttransfection. Lanes 1, 2, 3, and 4 contained EBNA2 Ser243E, EBNA2 Ser243D, wild-type EBNA2, and vector, respectively. Lamin B is used as a nuclear marker.

RNA protection assays (RPAs) show decreased LMP1 mRNA in EBNA2 Ser243D or Ser243E-transfected P3HR1 cells (Fig. 2B). Therefore, phosphorylation of Ser243 on EBNA2 can decrease its ability to induce LMP1 RNA consistent with an effect at the transcriptional level. This result is consistent with and confirms our previous findings that hyperphosphorylation of EBNA2 suppresses transactivation of the LMP1 promoter and produces a decreased steady-state level of LMP1 mRNA in cells in mitosis (46).

EBNA2 is a nuclear protein (10, 18). We compared the nuclear localization of wild-type EBNA2 and mutant EBNA2 proteins that are exogenously expressed in p3HR1 cells. As shown in Fig. 2C, the majority of EBNA2 protein resides in the nucleus (lane 1), and substitution of Ser243 with D or E (lanes 2 and 3) does not materially affect EBNA2 nuclear localization. These results indicate that the inhibitory effect on EBNA2 function is not due to change in nuclear localization caused by the substitutions.

To test whether phosphorylation at Ser243 has a general effect on EBNA2 transactivation function, we chose Cp as a prototype for study since EBNA2 transactivates Cp, the promoter for all of the EBNAs, as well as the LMP1 promoters. HeLa cells were chosen for the present study since transactivation of Cp by EBNA2 has been well studied in HeLa cells (17); more importantly, mitotic cells can be easily separated from interphase cells by the classical "shake-off" method (19, 42). First, transactivation of Cp by EBNA2 in M phase was decreased by ca. 50% compared to that in asynchronous cells (Fig. 3A). To rule out a non-cell-cycle-dependent effect of nocodazole, we used the irrelevant simian virus 40 early promoter as a control and found that its activity was not affected by nocodazole, similar to previous observations (46; data not shown).


Figure 3
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  		FIG. 3. Phosphorylation at Ser243 suppresses EBNA2 transactivation of the EBV BamHI C promoter, Cp. HeLa cells were cotransfected with reporter plasmid pLuc-Cp and pRL-TK, which encodes the Renilla luciferase gene in addition to empty vector or EBNA2 expression vector encoding wild-type EBNA2 (EBNA2 wt) or EBNA2 Ser243D. At 24 h after transfection, cells were arrested in M phase by nocodazole (M); untreated asynchronously growing cells served as a control (Asy). Luciferase assays were performed to evaluate promoter activity. In each phase, transactivation of Cp by EBNA2 is normalized to the vector control. (A) Decreased transactivation of Cp by EBNA2 in M phase. *, P < 0.01 versus control. (B) Comparison of transactivation of Cp by wild-type EBNA2, EBNA2 Ser243A and EBNA2 Ser243D. Each datum point (A and B) represents the average of three independent experiments done in triplicate. Error bars represent the means ± the standard deviation.

Second, we studied whether phosphorylation at Ser243 is involved in such suppression. As shown in Fig. 3B, transactivation of Cp by EBNA2 Ser243D is consistently lower than by wild-type EBNA2 (ca. 50% less), whereas EBNA2 Ser243A transactivation activity is similar to that of wild-type EBNA2. EBNA2 Ser243E has an effect similar to that of EBNA2 Ser243D (data not shown). These results suggest that phosphorylation at Ser243 is involved in suppression of EBNA2 transactivation of Cp.

Third, the endogenous mRNA levels of EBNA2 in asynchronously growing and mitotically arrested LCL-1 cells were compared. As shown in Fig. 4, in M phase the steady-state level of EBNA2 mRNA is ca. 56% of that detected in asynchronously growing cells. Transcription of all of the EBNA genes is driven by one of two promoters, Cp or Wp, and the activity of these promoters is mutually exclusive (39, 44, 45). Wp is used during the initial stages of B-cell immortalization, followed by a switch to Cp usage (39, 44, 45). In this experiment, transcription of EBNA2 from Cp but not Wp was confirmed by reverse transcription-PCR (21; data not shown). These and our previous (46) results indicate that activity of the two major EBV latency promoters LMP1p and Cp is suppressed in mitosis.


Figure 4
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  		FIG. 4. Decreased endogenous steady-state EBNA2 mRNA levels in M-phase-arrested cells. (A) RPA was performed on total RNA from asynchronously growing (Asy) and M-phase-arrested (M) LCL-1 cells with GAPDH and EBNA2 probe. Yeast RNA or total RNA from DG75 cells were used as negative controls. A representative result from three independent experiments is shown. (B) Relative mRNA levels of EBNA2 were analyzed by normalizing EBNA2 mRNA levels to the GAPDH mRNA level with a PhosphorImager. Each datum point represents the average of three independent experiments. Error bars represent the means ± standard deviation (*, P < 0.01 versus Asy control).

In addition to Ser243, there might be other sites phosphorylated on EBNA2 in mitosis, since migration of EBNA2 Ser243A from mitotic cells is still retarded compared to its counterpart in asynchronously growing cells (Fig. 1C). Although whether other such sites also participate in suppressing EBNA2 transactivation function is unknown, our data indicate that phosphorylation at Ser243 by itself is sufficient to suppress EBNA2's ability to induce endogenous LMP1 transcription in P3HR1 cells (Fig. 2A and B) and transactivate Cp (Fig. 3B), and therefore Ser243 is a principal site for regulation of EBNA2 function in mitosis. The results suggest that the suppression of EBNA2 transactivation of viral promoters in mitosis involves cdc2/cyclin B1 phosphorylation at Ser243.

Phosphorylation at Ser243 generally suppresses EBNA2 transactivation function most likely during mitosis, since the Ser243 site does not seem to be phosphorylated in S and G1 phase (Fig. 1C). In eukaryotic cells, entry into mitosis is accompanied by a global inhibition of transcription (34, 41), and this transcriptional repression is thought to be required for the accurate division of chromosomes during mitosis (reviewed in reference 13). Transcriptional repression in mitosis is regulated at different levels, among which phosphorylation of a series of transcriptional factors by a mitotic kinase, in most cases involved with cdc2/cyclin B1 kinase, is one of the important mechanisms (13). The behavior of EBV episomes resembles that of host cellular DNA in terms of being packaged in a nucleosomal structure, replicated once per cell cycle (12, 37), and partitioned at mitosis (7). We report here another similarity between viral episomal and host gene behavior during the cell cycle: suppression of viral transcription during mitosis, likely produced by phosphorylation with cdc2/cyclin B1 kinase. Although mitotic transcriptional suppression might not be apparent at the viral protein level, since EBV products including EBNA1, EBNA2, and LMP1 have longer half-lives (3, 4, 11, 14) than the duration of mitosis (ca. 1 h [2]), mitotic suppression of EBV episomal transcription might nonetheless have a biological impact similar to mitotic inhibition of cellular transcription machinery (13).

The EBNA2 construct used in the present study is type 1 EBNA2 from the B95-8 strain. Type 2 (AG876) EBNA2 (9), as well as several EBNA2 homologs from nonhuman primate lymphocryptoviruses (LCVs), including baboon LCV EBNA2 (28) and rhesus LCV EBNA2 (32), has been identified and sequenced. Interestingly, as predicted by Scansite (31), EBNA2 proteins from these other viruses also have putative cdc2 phosphorylation sites, which implies that EBNA2 homologs might also be substrates of cdc2 kinase. Although Ser243 is not conserved among EBNA2 homologs based on protein alignment (32), all have putative cdc2 phosphorylation sites within the region adjacent to Ser243 (approximately amino acids 230 to 250) (Fig. 5), suggesting that this region might be positionally conserved with regard to phosphorylation by cdc2 kinase. In fact, a positionally conserved region in EBNA2 for PKC phosphorylation has been reported (27). It is tempting to predict that similar mitotic transcriptional suppression of EBNA2-responsive genes might occur in type 2 EBV or LCV latently infected cells through cdc2 phosphorylation in this conserved region.


Figure 5
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  		FIG. 5. Alignment of the type 1 (B95-8), type 2 (AG876), baboon LCV and rhesus LCV EBNA2 amino acid sequences from amino acids 230 to 328. Clusters of amino acids CR5 and CR6 (32) that are conserved among all four EBNA2 proteins are indicated (boxed), as is the positionally conserved, cdc2 consensus phosphorylation motif (boxed). The putative cdc2 phosphorylation sites are marked with an asterisk.

Interestingly, Ser243 can also be phosphorylated by the EBV-encoded cytolytic cycle protein kinase (PK) (47). It is reasonable to deduce that phosphorylation at Ser243 by EBV PK might also have a general suppressive effect on EBNA2-responsive promoters. In LCLs generally transcriptional repression through Ser243 phosphorylation only happens in mitosis, whereas introducing EBV PK into EBV-transformed LCLs will theoretically repress EBNA2-responsive promoters constitutively and shut down the expression of latent gene products with eventual compromise of the type III latency state. Therefore, Ser243 is not only a key site during the entire EBV life cycle through which both cellular and viral kinases modulate EBNA2's transactivation function but also has potential utilities as a target for gene therapy (E. Gershburg, S. Kenney, and J. S. Pagano, unpublished data).


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ACKNOWLEDGMENTS
 
We thank Jeffrey Lin and Elliott Kieff for pLuc-Cp plasmid, Paul D. Ling for EBNA2 expression vector, and Matthew G. Davenport and Edward Gershburg for valuable discussion of the data.

This study was supported in part by a grant from the National Cancer Institute (CA 19014).


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FOOTNOTES
 
* Corresponding author. Mailing address: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Phone: (919) 966-5907. Fax: (919) 966-9673. E-mail: joseph_pagano{at}med.unc.edu. Back


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REFERENCES
 
    1
  1. Abbot, S. D., M. Rowe, K. Cadwallader, A. Ricksten, J. Gordon, F. Wang, L. Rymo, and A. B. Rickinson. 1990. Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein. J. Virol. 64:2126-2134.[Abstract/Free Full Text]
    2. 2
  2. Alberts, B. B. D., J. Lewis, M. Raff, K. Roberts, and J. D. Watson. 1989. Cell growth and division, p.729-790. In R. Adams and A. Walker (ed.), Molecular biology of the cell. Garland Publishing, Inc., New York, N.Y.
    3. 3
  3. Aviel, S., G. Winberg, M. Massucci, and A. Ciechanover. 2000. Degradation of the Epstein-Barr virus latent membrane protein 1 (LMP1) by the ubiquitin-proteasome pathway: targeting via ubiquitination of the N-terminal residue. J. Biol. Chem. 275:23491-23499.[Abstract/Free Full Text]
    4. 4
  4. Baichwal, V. R., and B. Sugden. 1987. Posttranslational processing of an Epstein-Barr virus-encoded membrane protein expressed in cells transformed by Epstein-Barr virus. J. Virol. 63:866-875.
    5. 5
  5. Bonnet, M., J. M. Guinebretiere, E. Kremmer, V. Grunewald, E. Benhamou, G. Contesso, and I. Joab. 1999. Detection of Epstein-Barr virus in invasive breast cancers. J. Natl. Cancer Inst. 91:1376-1381.[Abstract/Free Full Text]
    6. 6
  6. Bostock, C. J., D. M. Prescott, and J. B. Kirkpatrick. 1971. An evaluation of the double thymidine block for synchronizing mammalian cells at the G1-S border. Exp. Cell Res. 68:163-168.[CrossRef][Medline]
    7. 7
  7. Calos, M. P. 1998. Stability without a centromere. Proc. Natl. Acad. Sci. USA 95:4084-4085.[Free Full Text]
    8. 8
  8. Cohen, J. I., F. Wang, J. Mannick, and E. Kieff. 1989. Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation. Proc. Natl. Acad. Sci. USA 86:9558-9562.[Abstract/Free Full Text]
    9. 9
  9. Dambaugh, T., K. Hennessy, L. Chamnankit, and E. Kieff. 1984. U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2. Proc. Natl. Acad. Sci. USA 81:7632-7636.[Abstract/Free Full Text]
    10. 10
  10. Dambaugh, T., F. Wang, K. Hennessy, E. Woodland, A. Rickinson, and E. Kieff. 1986. Expression of the Epstein-Barr virus nuclear protein 2 in rodent cells. J. Virol. 59:453-462.[Abstract/Free Full Text]
    11. 11
  11. Davenport, M. G., and J. S. Pagano. 1999. Expression of EBNA-1 mRNA is regulated by cell cycle during Epstein-Barr virus type I latency. J. Virol. 73:3154-3161.[Abstract/Free Full Text]
    12. 12
  12. Dyson, P. J., and P. J. Farrell. 1985. Chromatin structure of Epstein-Barr virus. J. Gen. Virol. 66:1931-1940.[Abstract/Free Full Text]
    13. 13
  13. Gottesfeld, J. M., and D. J. Forbes. 1997. Mitotic repression of the transcriptional machinery. Trends Biochem. Sci. 22:197-202.[CrossRef][Medline]
    14. 14
  14. Grasser, F. A., P. Haiss, and N. Mueller-Lantzsch. 1991. Biochemical characterization of Epstein-Barr virus nuclear antigen 2A. J. Virol. 65:3779-3788.[Abstract/Free Full Text]
    15. 15
  15. Grinstein, S., M. V. Preciado, P. Gattuso, P. A. Chabay, W. H. Warren, E. De Matteo, and V. E. Gould. 2002. Demonstration of Epstein-Barr virus in carcinomas of various sites. Cancer Res. 62:4876-4878.[Abstract/Free Full Text]
    16. 16
  16. Harada, S., and E. Kieff. 1997. Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. J. Virol. 71:6611-6618.[Abstract]
    17. 17
  17. Henkel, T., P. D. Ling, S. D. Hayward, and M. G. Peterson. 1994. Mediation of Epstein-Barr virus EBNA2 transactivation by recombination signal-binding protein Jk. Science 265:92-95.[Abstract/Free Full Text]
    18. 18
  18. Hennessy, K., and E. Kieff. 1983. One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain. Proc. Natl. Acad. Sci. USA 80:5665-5669.[Abstract/Free Full Text]
    19. 19
  19. Jackman, J., and P. M. O'Commor. 1998. Cell cycle analysis, p. 8.0.1-8.0.13. In J. S. Bonifacino, M. Dasso, J. B. Harford, J. Lippincott-Schwartz, and K. M. Yamada (ed.), Current protocols in cell biology, vol. 1. John Wiley & Sons, Inc., New York, N.Y.
    20. 20
  20. Johannsen, E., E. Koh, G. Mosialos, X. Tong, E. Kieff, and S. R. Grossman. 1995. Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J{kappa} and PU. 1. J. Virol. 69:253-262.[Abstract]
    21. 21
  21. Kelly, G., A. Bell, and A. Rickinson. 2002. Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2. Nat. Med. 8:1098-1104.[CrossRef][Medline]
    22. 22
  22. Kwiatkowski, B., S. Y. J. Chen, and W. H. Schubach. 2004. CKII site in Epstein-Barr virus nuclear protein 2 controls binding to hSNF5/Ini1 and is important for growth transformation. J. Virol. 78:6067-6072.[Abstract/Free Full Text]
    23. 23
  23. Lee, J.-S., K. M. Collins, A. L. Brown, C.-H. Lee, and J. H. Chung. 2000. hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response. Nature 404:201-204.[CrossRef][Medline]
    24. 24
  24. Lee, S. Y., M. R. Wenk, Y. Kim, A. C. Nairn, and P. De Camilli. 2004. Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses. Proc. Natl. Acad. Sci. USA 101:546-551.[Abstract/Free Full Text]
    25. 25
  25. Leibold, W., T. D. Flanagan, J. Menezes, and G. Klein. 1975. Induction of Epstein-Barr virus-associated nuclear antigen during in vitro transformation of human lymphoid cells. J. Natl. Cancer Inst. 54:65-68.[Medline]
    26. 26
  26. Lindahl, T., A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn. 1976. Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. J. Mol. Biol. 102:511-530.[CrossRef][Medline]
    27. 27
  27. Ling, P. D., and S. D. Hayward. 1995. Contribution of conserved amino acids in mediating the interaction between EBNA2 and CBF1/RBPJk. J. Virol. 69:1944-1950.[Abstract]
    28. 28
  28. Ling, P. D., J. J. Ryon, and S. D. Hayward. 1993. EBNA-2 of herpesvirus papio diverges significantly from the type A and type B EBNA-2 proteins of Epstein-Barr virus but retains an efficient transactivation domain with a conserved hydrophobic motif. J. Virol. 67:2990-3003.[Abstract/Free Full Text]
    29. 29
  29. Moreno, S., and P. Nurse. 1990. Substrates for p34 cdc2: in vivo veritas? Cell 61:549-551.[CrossRef][Medline]
    30. 30
  30. Ning, S., A. M. Hahn, L. E. Huye, and J. S. Pagano. 2003. Interferon regulatory factor 7 regulates expression of Epstein-Barr virus latent membrane protein 1: a regulatory circuit. J. Virol. 77:9359-9368.[Abstract/Free Full Text]
    31. 31
  31. Obenauer, J. C., L. C. Cantley, and M. B. Yaffe. 2003. Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 31:3635-3641.[Abstract/Free Full Text]
    32. 32
  32. Peng, R., A. V. Gordadze, E. M. Fuentes Panana, F. Wang, J. Zong, G. S. Hayward, J. Tan, and P. D. Ling. 2000. Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses. J. Virol. 74:379-389.[Abstract/Free Full Text]
    33. 33
  33. Pope, J. H. 1967. Establishment of cell lines from peripheral leucocytes in infectious mononucleosis. Nature 216:810-811.[CrossRef][Medline]
    34. 34
  34. Prescott, D. M., and M. A. Bender. 1962. Synthesis of RNA and protein during mitosis in mammalian tissue culture cells. Exp. Cell Res. 26:260-268.[CrossRef][Medline]
    35. 35
  35. Rayet, B., Y. Fan, and C. Gelinas. 2003. Mutations in the v-Rel transactivation domain indicate altered phosphorylation and identify a subset of NF-{kappa}B-regulated cell death inhibitors important for v-Rel transforming activity. Mol. Cell. Biol. 23:1520-1533.[Abstract/Free Full Text]
    36. 36
  36. Rickinson, A. B., and E. Kieff. 2001. Epstein-Barr virus, p. 2575-2627. In D. M. Knipe and P. M. Howley (ed.), Fields virology. Lippincott, Philadelphia, Pa.
    37. 37
  37. Sexton, C. J., and J. S. Pagano. 1989. Analysis of the Epstein-Barr virus origin of plasmid replication (oriP) reveals an area of nucleosome sparing that spans the 3' dyad. J. Virol. 63:5505-5508.[Abstract/Free Full Text]
    38. 38
  38. Shaw, J. E., L. F. Levinger, and C. W., Jr. Carter. 1979. Nucleosomal structure of Epstein-Barr virus DNA in transformed cell lines. J. Virol. 29:657-665.[Abstract/Free Full Text]
    39. 39
  39. Speck, S. H., and J. L. Strominger. 1989. Transcription of Epstein-Barr virus in latently infected growth-transformed lymphocytes. Adv. Viral. Oncol. 8:133-150.
    40. 40
  40. Sung, N. S., S. Kenney, D. Gutsch, and J. S. Pagano. 1991. EBNA-2 transactivates a lymphoid-specific enhancer in the Bam H1 C promoter of Epstein-Barr virus. J. Virol. 65:2164-2169.[Abstract/Free Full Text]
    41. 41
  41. Taylor, J. H. 1960. Nucleic acid synthesis in relation to the cell division cycle. Ann. N. Y. Acad. Sci. 90:409-421.[CrossRef][Medline]
    42. 42
  42. Terasima, T., and L. J. Tolmach. 1963. Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells. Exp. Cell Res. 30:344-362.[CrossRef][Medline]
    43. 43
  43. Wang, F., S. F. Tsang, M. G. Kurilla, J. I. Cohen, and E. Kieff. 1990. Epstein-Bar virus nuclear antigen 2 transactivates latent membrane protein LMP-1. J. Virol. 64:3407-3416.[Abstract/Free Full Text]
    44. 44
  44. Woisetschlaeger, M., J. L. Strominger, and S. H. Speck. 1989. Mutually exclusive use of viral promoters in Epstein-Barr virus latently infected lymphocytes. Proc. Natl. Acad. Sci. USA 86:6498-6502.[Abstract/Free Full Text]
    45. 45
  45. Woisetschlaeger, M., C. N. Yandava, L. A. Furmanski, J. L. Strominger, and S. H. Speck. 1990. Promoter switching in Epstein-Barr virus during the initial stages of infection of B lymphocytes. Proc. Natl. Acad. Sci. USA 87:1725-1729.[Abstract/Free Full Text]
    46. 46
  46. Yue, W., M. G. Davenport, J. Shackelford, and J. S. Pagano. 2004. Mitosis-specific hyperphosphorylation of Epstein-Barr virus nuclear antigen 2 suppresses its function. J. Virol. 78:3542-3552.[Abstract/Free Full Text]
    47. 47
  47. Yue, W., E. Gershburg, and J. S. Pagano. 2005. Hyperphosphorylation of EBNA2 by Epstein-Barr virus protein kinase suppresses transactivation of the LMP1 promoter. J. Virol. 79:5880-5885.[Abstract/Free Full Text]
    48. 48
  48. Zimber-Strobl, U., E. Kremmer, F. Grasser, G. Marschall, G. Laux, and G. W. Bornkamm. 1993. The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA-2 responsive cis-element of the terminal protein 1 gene promoter. EMBO J. 12:167-175.[Medline]
    49. 49
  49. Zimber-Strobl, U., K. O. Suentzenich, G. Laux, D. Eick, M. Cordier, A. Calender, M. Billaud, G. M. Lenoir, and G. W. Bornkamm. 1991. Epstein-Barr virus nuclear antigen 2 activates transcription of the terminal protein gene. J. Virol. 65:415-423.[Abstract/Free Full Text]

Journal of Virology, February 2006, p. 2045-2050, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.2045-2050.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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