cdc2/Cyclin B1-Dependent Phosphorylation of EBNA2
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病菌学杂志 2006年第4期
Department of Medicine
Department of Microbiology and Immunology
Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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.
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.
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. -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.
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).
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.
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.
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).
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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Department of Microbiology and Immunology
Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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.
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.
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. -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.
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).
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.
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.
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).
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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