Epstein-Barr Virus BZLF1 Protein Binds to Mitotic Chromosomes

Transcription

1 JOURNAL OF VIROLOGY, June 2005, p Vol. 79, No X/05/$ doi: /jvi Copyright 2005, American Society for Microbiology. All Rights Reserved. Epstein-Barr Virus BZLF1 Protein Binds to Mitotic Chromosomes Amy L. Adamson* Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina Received 3 November 2004/Accepted 14 February 2005 Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and is associated with several types of cancers, including nasopharyngeal carcinoma and Burkitt s lymphoma. An EBV protein that plays an integral role during lytic replication is the immediate-early protein BZLF1. Our laboratory has found that BZLF1 (Z) localizes to host chromosomes during mitosis. Two Z-interacting proteins are also found localized to mitotic chromosomes in the presence of Z. The association between Z and mitotic chromosomes may lead to the sequestering of Z-interacting proteins within the cell and potentially cause an alteration of chromosome compaction or chromatin structure. Epstein-Barr virus (EBV) is a human herpesvirus that has infected about 90% of the world s population. If primary infection with EBV occurs in adolescents or adults, it causes infectious mononucleosis, a typically short-lived but acute syndrome that can render complications in internal organs such as the liver (33, 43). EBV has also been implicated in a variety of cancers, including Burkitt s lymphoma and nasopharyngeal carcinoma (NPC). These cancers may be linked to certain genetic or environmental conditions (33). During primary infection, EBV perpetuates itself in a lytic (productive) manner, where the majority of EBV genes are expressed, in order to reproduce the virus. Infection of B cells can occur during this time, which leads to the immortalization of a subset of these B cells, producing a permanent shelter for the EBV genome (24, 33). To trigger EBV lytic replication, two EBV genes, coding for the proteins BZLF1 and BRLF1, are expressed (8, 10, 23, 35, 36, 39, 41). These immediate-early genes encode transcriptional activators that bind to and activate EBV early gene promoters (8, 11, 14, 15, 17, 22, 32). BZLF1 (Z) is a bzip protein, and its DNA binding domain bears homology to the AP1 site binding proteins, c-jun and c-fos (12). Therefore, Z is able to bind to AP1 and AP1-like sites, which are present in the promoters of the EBV early genes (24, 40). The Z protein also has domains required for transcriptional activation, viral replication, and protein dimerization (7, 13, 18, 25, 26, 31, 37). Besides playing a major role in EBV viral replication, Z and BRLF1 have been shown to affect a variety of nonviral cellular protein functions and pathways. Z is SUMO-1 (small ubiquitin-related modifier 1) modified (1), which has been shown, for other proteins, to alter protein activity. Z physically interacts with several important regulatory proteins, including p53, p65, and CBP (CREB-binding protein) (2, 16, 42). Such interactions likely advocate viral replication and survival, while potentially harming the normal cell state. In addition, BRLF1 overexpression has been shown to activate cell cycle progression (38), while Z overexpression has been shown to arrest the cell cycle either in G 0 /G 1 or G 2 /M, depending upon the cell * Mailing address: Department of Biology, University of North Carolina at Greensboro, Greensboro, NC Phone: (336) Fax: (336) aladamso@uncg.edu. type under study (5, 6, 29). However, in the context of Z expression from the endogenous EBV genome (in EBV-positive cells), the cell cycle effects are not so clear. Rodriguez et al. showed that the induction of lytic replication in a variety of EBV-positive cells had different cell cycle profiles for the cells that expressed Z (34). While NPC-KT and P3HR1 cells appeared to have a G 1 /G 0 arrest, Rael cells appeared to have a G 2 /M arrest, and Akata cells had no cell cycle arrest at all. It is noteworthy to mention that all of these cell cycle profiles still included 14 to 30% cells in S phase (34). Mauser et al. found that in the AGS-EBV cell line, cells that constitutively expressed Z without induction actually had more cells in S phase than the non-z-expressing cells (28). This indicates that the effects of EBV, and specifically Z, on the cell cycle vary and that the expression of Z in cells does not necessarily stop cells from entering and going through mitosis. During mitosis, chromosomes become tightly compacted. DNA is initially compacted into nucleosomes by histone proteins and further compacted by scaffolding proteins and the condensin protein, which wraps DNA into supercoils. The complex of DNA, histones, and nonhistone proteins excludes most transcription factor binding. The result is that transcription is generally repressed during mitosis (19, 21). Z binds to mitotic chromosomes. Z is a nuclear protein. In order to examine where in the cell Z was found during mitosis, HeLa cells (from American Type Culture Collection) were transfected with a Z expression vector (SvpIE-Z; contains genomic Z DNA) and subsequently stained with an anti-z antibody (from Argene) and Hoechst stain (which stains DNA; from Sigma). Interestingly, we found cells that were positively stained for Z, in mitosis, with Z protein localized to the mitotic chromosomes (Fig. 1). This finding was not due to bleedthrough of the Hoechst stain into the anti-z antibody channel, as demonstrated in Fig. 1A and B. Notably, Z localized to mitotic chromosomes during prophase, metaphase, and anaphase (Fig. 1C to H). During mitosis, the Z protein appeared to be mostly confined to the chromosomes, with little staining elsewhere in the cell. This is in contrast to the localization of Z protein in interphase cells, which appears to be evenly spread throughout the nucleus (Fig. 1). To verify that the Z/chromosomal staining was not an artifact due to the transfection process, we transfected into HeLa cells several expression con- 7899

2 7900 NOTES J. VIROL. FIG. 1. Z localizes to mitotic chromosomes. HeLa cells were transfected with a Z expression plasmid and stained with Hoechst stain (A, C, E, G) and an anti-z antibody (B, D, F, H). Four different fields of cells are shown (A-B, C-D, E-F, G-H). Asterisks indicate Z-containing cells. Panels A and B serve as a control to show that the Hoechst stain does not bleed-through into the anti-z channel. structs for other EBV and cellular proteins and found that none of these other proteins bound to mitotic chromosomes (data not shown). In addition, the anti-z antibody specifically detected Z protein on mitotic chromosomes and did not crossreact with the mitotic DNA alone, since untransfected HeLa cells that were immunostained with the anti-z antibody did not yield any signal (data not shown). Since HeLa cells are infected with human papillomavirus and thus express other viral proteins, we transfected the Z expression vector into the mouse fibroblast cell line NIH 3T3 (from American Type Culture Collection) to test for mitotic chromosome localization. We found that, just as in HeLa cells, Z protein localized to chromosomes in mitotic cells (Fig. 2A and B). To examine whether Z would bind to host chromosomes in the presence of the EBV viral genome, we transfected the Z expression vector into the EBV latently infected D98-HE/R1 cell line (from Shannon Kenney). Staining with an anti-z antibody revealed the same localization as in HeLa and NIH 3T3 cells; that is, Z bound to the host chromosomes in the presence of the EBV genome and other EBV proteins (Fig. 2C and D). Endogenous BZLF1 binds to mitotic chromosomes. Since the Z localization to mitotic chromosomes that we have demonstrated has been shown in cells transfected with a Z expression vector, we next investigated whether Z expressed from the FIG. 2. Z localizes to mitotic chromosomes in NIH 3T3 cells and D98-HE/R1 cells. NIH 3T3 cells were transfected with a Z expression plasmid and stained with Hoechst stain (A) and an anti-z antibody (B). D98-HE/R1 cells were transfected with a Z expression plasmid and stained with Hoechst stain (C) and an anti-z antibody (D). Arrows indicate mitotic cells. endogenous viral genome would show the same localization. To induce lytic replication in D98-HE/R1 cells, we transfected the cells with an expression vector for the immediate-early gene coding for BRLF1. BRLF1, like Z, is capable of disrupting viral latency and activates the Z promoter. Forty-eight hours posttransfection, we immunostained the cells with the anti-z antibody. As in the other cell types previously shown, the induced Z protein bound to mitotic chromosomes (Fig. 3). Therefore, the endogenous EBV Z protein was able to bind to chromosomes and did so in the midst of lytic replication. BZLF1 brings CBP and Pax5 to mitotic chromosomes. Z interacts with several cellular proteins. Therefore, we examined whether Z would continue to interact with a binding partner while also bound to mitotic chromosomes. We have previously demonstrated a physical interaction between Z and CBP (CREB-binding protein) (2). CBP is an acetyltransferase that acts as a transcriptional coactivator. In untransfected HeLa cells, CBP is present spread throughout the nucleus (data not shown). To examine the localization of CBP in Z- expressing cells, we immunostained HeLa cells that had been transfected with a Z expression vector with anti-z and anti- CBP (from Upstate Biotechnology) antibodies. In cells that did not express Z, we found that, during interphase, CBP was generally evenly spread throughout the nucleus, with some brighter-staining dots present (Fig. 4C). In mitosis, CBP was also found spread throughout the cell (Fig. 4C). However, the localization of CBP was altered in cells that expressed Z, such that the CBP was found on the mitotic chromosomes with Z, instead of being evenly spread throughout the cell (Fig. 4F). We have previously demonstrated a physical interaction between Z and Pax5 (unpublished data), a human transcription factor that is necessary for B-cell differentiation (30). When an expression vector for Pax5 was transfected into HeLa cells, Pax5 did not localize to mitotic chromosomes (Fig. 5B) (anti-

3 VOL. 79, 2005 NOTES 7901 FIG. 3. Cells induced into the lytic cycle have BZLF1 on mitotic chromosomes. Latently infected D98/HE-R1 cells were transfected with an expression vector for BRLF1 to trigger lytic replication. Cells were immunostained for Z (B, D). The DNA was stained with Hoechst stain (A, C). Two representative cells are shown, showing both anaphase (A, B) and metaphase (C, D) chromosomes. Arrows refer to mitotic cells. Pax5 antibody was from Santa Cruz). However, when Pax5 was expressed in conjunction with Z, Pax5 did localize to mitotic chromosomes, along with Z (Fig. 5D). This suggests that Z continues to bind to other proteins while associated with chromosomes and may be able to sequester cellular proteins on chromosomes, even when such cellular proteins do not normally bind to chromosomes during mitosis. To ensure that the Z/Pax5 and Z/CBP colocalizations that we saw on mitotic chromosomes were not due to cross-reactivity of the antibodies with chromatin, we immunostained untransfected HeLa cells with anti-z, anti-pax5, or anti-cbp antibodies. We did not detect any antibody reaction to chromosomes in mitotic cells (data not shown). Therefore, the colocalizations that we have demonstrated are specific and not due to cross-reactivity of the antibodies used. To investigate whether Z was able to translocate endogenous Pax5 protein to mitotic chromosomes, we transfected Raji cells (B cells latently infected with EBV; from Shannon Kenney) with the Z expression vector and immunostained these cells with anti-z and anti-pax5 antibodies. While not all of the cells expressed endogenous Pax5, we were able to find many cells in which both Z and Pax5 proteins were present. In interphase cells, Z protein was often found in discrete compartments within the nucleus, and Pax5 protein colocalized with Z in these compartments (Fig. 6A to C). In mitotic cells, however, Z protein was localized to the host chromosomes and Pax5 colocalized with Z on these chromosomes (Fig. 6D to F). In mitotic cells that had been transfected with a control vector, the endogenous Pax5 protein did not localize to chromosomes (Fig. 6G and H). Therefore, Z is able to translocate endogenous Pax5 to chromosomes during mitosis. The DNA binding domain of BZLF1 is necessary for binding to chromosomes. Since Z is a DNA binding protein that can bind to AP1 and AP1-like sites, it was reasonable that Z associated with chromosomes through its DNA binding domain. Alternatively, Z may have bound to chromosomes through an indirect, protein-protein interaction. To test whether the DNA binding domain of Z was necessary for the localization, we transfected HeLa cells with an expression vector for a Z mu- FIG. 4. Z directs CBP to mitotic chromosomes. HeLa cells were transfected with an expression vector for Z. Cells were stained with Hoechst stain (A, D), an anti-z antibody (B, E), and an anti-cbp antibody (C, F). Compare the localization of CBP in mitotic cells either containing Z (F) or not containing Z (C). The confocal images were taken sequentially to ensure that there was no bleed-through between channels. M, mitotic cell; I, interphase cell.

4 7902 NOTES J. VIROL. FIG. 5. Z directs Pax5 to mitotic chromosomes. HeLa cells were transfected with expression vectors for Pax5 alone (A, B) or Pax5 plus Z (C, D, E). Cells were stained with Hoechst stain (A, C), an anti-pax5 antibody alone (B), or anti-pax5 plus anti-z antibodies together (D, E). Asterisks represent transfected cells. Compare localization of Pax5 in the mitotic cells of panel B versus D. Downloaded from tant, Z311, that cannot bind DNA (Z311 has an alteration of amino acid 185, from alanine to lysine; from Shannon Kenney). Figure 7 shows that Z311 was unable to bind to chromosomes and remained dispersed throughout the cell during mitosis (Fig. 7D). Even though the DNA binding domain of Z appears to be required for chromosome localization, this does not preclude other regions of Z from playing a role in this interaction. Z increases the level of acetylated histone H3 on mitotic chromosomes. CBP is a histone acetylase that acts to acetylate histones in chromatin. Since we found that Z translocates CBP to mitotic chromosomes during mitosis (Fig. 4), we sought to examine whether there was a change in the acetylation status of histones in mitotic chromosomes when Z was bound to these chromosomes. To this end, we transfected HeLa cells with either a control vector or Z expression vector and subsequently immunostained these cells with anti-z and anti-acetylated histone H3 (from Abcam) antibodies, as well as the Hoechst DNA stain (Fig. 8). To analyze the levels of acetylated histone H3 on mitotic chromosomes, we quantitated the relative intensities of acetylated histone H3 staining and DNA staining, and for each set of mitotic chromosomes, we calculated the ratio between these two intensities. The averages of these ratios are presented in Table 1. We found that there was a significantly higher ratio of acetylated histone H3 to DNA for the mitotic chromosomes that were bound by Z in comparison to the ratio for the mitotic chromosomes in control cells FIG. 6. Z binds to chromosomes in B cells and translocates endogenous Pax5 to chromosomes. Raji cells were transfected with a Z expression vector (A to F) or a control vector (G, H) and immunostained with Hoechst stain (A, D, G), an anti-pax5 antibody (B, E, H), and an anti-z antibody (C, F). A representative interphase cell is shown in panels A to C, and a mitotic cell is shown in panels D to F. A mitotic cell that expresses Pax5 only is shown in panels G and H. Brackets indicate one cell. on June 27, 2018 by guest

5 VOL. 79, 2005 NOTES 7903 TABLE 1. Z increases the level of acetylated histone H3 on mitotic chromosomes a Protein n Mean intensity DNA Acetyl-H3 Mean ratio SD Vector control , , Z , , a n is the number of individual mitotic cells tested. The mean DNA and acetyl-h3 (acetylated histone H3) intensities are the relative intensity units per mitotic cell. The mean ratio is the average of the ratios of acetylated histone H3 to DNA on mitotic chromosomes. t 21, ; P FIG. 7. Z311 does not bind to mitotic chromosomes. HeLa cells were transfected with expression vectors for Z (A, B) or the DNA binding-defective Z311 (C, D). Cells were stained with Hoechst stain (A, C) and an anti-z antibody (B, D). Asterisks denote transfected cells. Arrows indicate mitotic cells. (using a confidence level of P 0.05, our t statistic was 5.2 with 21 degrees of freedom). These results suggest that when Z translocates cellular proteins such as CBP to mitotic chromosomes, these proteins remain functional and can significantly alter the normal structure of the chromosomes. Our results indicate that the EBV Z protein directly interacts with mitotic chromosomes. Both exogenous Z and endogenous Z are capable of this interaction. During this localization, Z continues to interact with at least two of its known binding partners, which then also localize to mitotic chromosomes. Z binding to chromosomes was observed in a variety of cell types, including epithelial, fibroblast, and B cells. So why does Z bind to mitotic chromosomes? The interaction may facilitate an equal distribution of Z protein to daughter cells. Alternatively, since it is known that the herpesvirus 8 LANA 1 protein and the EBV EBNA1 protein both bind to mitotic chromosomes and seemingly link viral replication and segregation during the cell cycle (3, 4, 9), Z may also play a role in segregating replicating EBV genomes in mitotic cells. Apart from of the purpose of the Z-chromosome interactions, the end result is that Z protein, as well as other Z-interacting proteins, binds to mitotic chromosomes and will consequently affect mitotic chromosome architecture. Most transcription factors are excluded from DNA during mitosis, in order for proper DNA compaction to occur. The binding of Z to DNA could potentially prevent full compaction of chromosomes and could lead to mitotic arrest. Mauser et al. have in fact shown that Z-expressing cells contain undercondensed mitotic chromosomes (29), which correlates with this theory. In addition, the proteins that Z brings to chromosomes may function to modify the chromatin. We have shown that there is a significant increase in histone H3 acetylation when Z is bound to mitotic chromosomes, presumably via the CBP that Z has tethered to the chromosomes. This may contribute to the undercondensed phenotype of these mitotic chromosomes and may also affect transcriptional regulation in these cells. In regard to Pax5, Johnson et al. have shown that Pax5 is necessary and sufficient for demethylation of lysine 9 on histone H3, thus allowing V(H)-to-DJ(H) recombination (20). Demethylation of the lysine allows the recombinase machinery access to the DNA (20). Therefore, when Pax5 is tethered to chromatin via Z, Pax5 may be able to demethylate the H3 lysine 9, resulting in chromatin modifications. These effects on chromosomes by Z are important considering that, in some tumor cells, such as in NPC, Z may be expressed but not evoke a complete lytic cycle (27). Therefore, Z may be expressed in cells that will not lyse to release viral particles, and Z s effects on chromosome stability could contribute to tumorigenesis. We have shown that the Z protein has a strong affinity for mitotic chromosome binding and that Z can alter the localization of cellular proteins during this process. This reorganization of protein binding may have a major impact upon chromosome condensation, chromatin structure, and normal cell function. FIG. 8. Z increases the level of acetylated histone H3 on mitotic chromosomes. HeLa cells were transfected with a Z expression vector (A to C) or a control vector (D, E). Cells were stained with Hoechst stain (A, D), an anti-acetylated histone H3 antibody (B, E), and an anti-z antibody (C). A confocal microscope was used to capture images, using the same exposure settings for all cells. REFERENCES 1. Adamson, A. L., and S. Kenney Epstein-Barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies. J. Virol. 75: Adamson, A. L., and S. Kenney The Epstein-Barr virus BZLF1 protein interacts physically and functionally with the histone acetylase CREB-binding protein. J. Virol. 73: Ballestas, M. E., P. A. Chatis, and K. M. Kaye Efficient persistence of

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