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Originally published In Press as doi:10.1074/jbc.M405911200 on October 21, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54817-54825, December 24, 2004
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In Vivo Dynamics of EBNA1-oriP Interaction during Latent and Lytic Replication of Epstein-Barr Virus*

Tohru Daikoku{ddagger}§, Ayumi Kudoh{ddagger}§, Masatoshi Fujita¶, Yutaka Sugaya{ddagger}, Hiroki Isomura{ddagger}, and Tatsuya Tsurumi{ddagger}||

From the {ddagger}Division of Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan and the Virology Division, National Cancer Center, Chuo-ku, Tokyo 104-0045, Japan

Received for publication, May 27, 2004 , and in revised form, September 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Epstein-Barr virus (EBV)1 is a human herpes virus that infects 90% of individuals. Primary EBV infection targets resting B lymphocytes, inducing their continuous proliferation. In the B lymphoblastoid cell lines, only limited numbers of viral genes are usually expressed and there is no production of virus particles, this being called latent infection. In the latent state, EBV maintains its 170-kbp genome as complete, multiple copies of plasmids. Latent phase viral replication appears to faithfully mimic cellular replicons; the EBV genomes or small EBNA1-oriP plasmids are synthesized only once in each S-phase by the host cell replication machinery, following the rules of chromosome replication (1).

EBV-infected cell lines usually contain a small subpopulation of cells that have switched spontaneously from a latent stage of infection into the lytic cycle. The mechanism of switching is not fully understood, but one of the first detectable changes is expression of the BZLF1 gene product. The BZLF1 protein, together with the protein product of the BRLF1 gene, transactivates viral and certain cellular promoters (2) and leads to an ordered cascade of viral gene expression. The lytic phase of EBV DNA replication is dependent on seven viral replication proteins: BZLF1, an oriLyt-binding protein; BALF5, a DNA polymerase; BMRF1, a polymerase processivity factor; BALF2, a single-stranded DNA-binding protein; and BBLF4, BSLF1, and BBLF2/3, predicted to be helicase-, primase-, and helicase-primase-associated proteins, respectively (3). All of these proteins except for BZLF1 conceivably work together at replication forks to synthesize leading and lagging strands of the concatemeric EBV genome (4). Interestingly, viral lytic replication occurs in discrete sites in nuclei called replication compartments in which viral replication proteins are assembled as we demonstrated.2

The EBNA1 protein is the only EBV-encoded protein required for the replication, retention, and partition of plasmid DNA during the latent phase of EBV replication (5, 6). It is detected in all types of EBV-infected cells and binds directly to the latent replication origin region, oriP, of EBV DNA as a homodimer (7). The oriP replicon differs from other viral replicons in that EBNA1 does not have ATPase or helicase activity (7, 8). oriP consists of two sequence elements, the family of repeats (FR) element and the dyad symmetry (DS) element, separated by a stretch of ~960 base pairs. FR contains a 20x 30-bp repeat arranged in tandem, each containing an EBNA1 binding site (9, 10). In EBNA1-expressing cells, FR is essential for the stable maintenance of EBV plasmids in nuclei (11). This element contains the termination site for replication (12, 13) and also works as a replication enhancer (14). DS contains four binding sites for EBNA1 with lower affinity than those in FR, two of which are part of a 65-bp dyad (9, 10, 15, 16). In EBNA1-expressing cells, DS most likely functions as the physical origin of bidirectional replication of EBV-derived plasmids (13, 14, 17). EBNA1 has also been observed to mediate efficient interaction at a distance between the FR and DS elements in vitro and to distort the structure of the DS element DNA both in vitro and in vivo. This interaction results in the generation of looped DNA molecules when the interaction occurs between EBNA1 bound to FR and DS elements in the same DNA molecule (8). Another mechanism by which EBNA1 could contribute to oriP replication would be for it to associate with the origin recognition complex (18) and tether it to DS at a site near or at which DNA synthesis from oriP initiates (13). Furthermore, EBNA1 binds to the metaphase chromosomes (19–21). This binding is considered to facilitate the partition of a low copy number of latent EBV plasmids that replicate once per cell cycle into dividing cells (20). On the other hand, EBNA1 bound to FR can activate transcription of the EBV BamHI-C and latent membrane protein-1 promoters, one of which is 10 kbp distant (22, 23). Thus, the ability of EBNA1 to bind to DNA is essential for its activation of replication and transcription through oriP. After induction of the lytic program, EBNA1 transcripts are still synthesized from the F promoter in place of the C promoter (24, 25), suggesting an unknown function in lytic infection.

We have previously isolated EBV latently infected Tet-BZLF1/B95-8 cells in which exogenous BZLF1 protein is conditionally expressed under the control of a tetracycline-regulated promoter (26). More than 80% of Tet-BZLF1/B95-8 cells become positive for BZLF1 and BMRF1 proteins 24 h after doxycycline annexation. The copy number of the viral genome in the cells is significantly amplified after 24 h post induction, and extracellular virus particles are produced. Thus, the system is simple and highly efficient for the conditional induction of the lytic replication program in the absence of any other external stimuli. Using this system, we investigated the in vivo dynamics of EBNA1-oriP interaction during latent and lytic replication. Biochemical fractionation and an EBV genome-wide chromatin immunoprecipitation (ChIP) assay clearly demonstrated that EBNA1 tightly binds to the oriP region in the entire EBV genome throughout the cell cycle during latent phase. We found that although the total levels of EBNA1 were constant throughout the lytic infection, levels of the DNA-bound form increased as lytic replication proceeded. The ChIP assay indicated that after the induction of lytic replication, EBNA1 still bound to the oriP region of the viral genomes. Furthermore, confocal laser microscopy analyses revealed that EBNA1 became redistributed to the viral replication compartments and localized as distinct spots within and/or juxtaposed to the compartments during lytic replication. Understanding the dynamics of EBNA1-DNA interactions in vivo during lytic replication should shed further light on EBNA1 functions in the EBV life cycle.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—B95-8 B lymphoblastoid cell line and Burkitt's cell lines Raji, Daudi, and Akata were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2. Tet-BZLF1/B95-8 cells were similarly maintained in RPMI 1640 medium supplemented with 1 µg/ml puromycin, 250 µg/ml hygromycin B, and 10% tetracycline-free fetal calf serum (Clontech) at 37 °C in a humidified atmosphere containing 5% CO2. To induce lytic EBV replication, the tetracycline derivative doxycycline was added to the culture medium at a final concentration of 2 µg/ml.

Plasmids—The EBV B95-8 genome library constructed from BamHI restriction enzyme-digested fragments of the B95-8 genome into pBR322 (27) was used for an EBV genome-wide ChIP assay. Plasmid pKORI, an oriP plasmid containing both FR and DS elements (EcoRI(7,315)-SacII(9516)), and plasmid pKS, containing an FR region (EcoRI(7,315)-EcoRV(8,992)), were gifts from Dr. Shirakata (28). The plasmids pBamC-1, pBamC-2, pBamC-3, and pBamC-4 were constructed by cloning the DNA fragments BamHI(3,994)-SacI(6281), SacI(6,281)-EcoRI(7,315), KpnI(10,308)-SacI(11,084), and SacI(11,084)-BamHI(13,215), respectively, into the appropriate multiple cloning sites of the pUC18 vector.

Antibodies—Anti-EBNA1 rabbit polyclonal antibodies were prepared as follows. A recombinant EBNA1 protein expressed in sf21 insect cells by infecting an AcEBNA1 recombinant baculovirus (7) was purified. Rabbits were injected with ~200 µg of the purified protein mixed with the RIBI adjuvant system R-730 (RIBI Immunochem Research), and anti-EBNA1 antibodies were affinity-purified from the antiserum as described (29). With Western blotting, the anti-EBNA1 antibody recognized a protein migrating at 72 kDa in whole cell lysates from B95-8 cells, the mobility of which, upon SDS-PAGE, was as expected for EBNA1 (data not shown). The antibodies also could immunoprecipitate the 72-kDa protein, which was thus concluded to be EBNA1 (data not shown). The antibody recognized a 66-kDa protein in a Raji cell extract (Fig. 2A).



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  		FIG. 2.
Biochemical analyses of the localization of EBNA1 in a lytic program induced Tet-BZLF1/B95-8 cells. A, expression levels of the EBNA1 protein in EBV-latently infected B cell lines. Whole cell lysates of Akata, B95-8, Tet-BZLF1/B95-8, Daudi, and Raji cells (corresponding to 1.8 x 104 cells) were applied to each lane and analyzed for expression levels of EBNA1 by immunoblotting with an anti-EBNA1 antibody. B, expression profiles of the EBNA1 protein during lytic replication. At the indicated times after induction, whole cell lysates of Tet-BZLF1/B95-8 cells were prepared and analyzed for expression levels of EBNA1 by immunoblotting with the anti-EBNA1 antibody. C, lytic replication was induced in Tet-BZLF1/B95-8 cells with 2 µg/ml doxycycline, and cells were harvested at 0, 24, 48, or 72 h post induction. Whole cell lysate (W), Triton X-100-extractable supernatants (S) and extracted nuclear pellets (P) were prepared as described in the legend to Fig. 1. The extracted nuclei were further treated with DNase I to obtain DNase I-solubilized supernatants containing chromatin and EBV genomes (S), and the remainder were insoluble non-chromatin nuclear pellets (DNase I, P). These fractions were analyzed by immunoblotting with an anti-EBNA1 antibody. Samples corresponding to 1.8 x 104 cells were applied to each lane. Tet-BZLF1/B95-8 cells were also cultured in the presence of PAA (400 µg/ml) and processed similarly. D, augmentation of DNA-bound form of EBNA1 as lytic replication proceeds. EBNA1 was detected with enhanced chemiluminescence reagents. Images were processed by LumiVisionPRO (Aisin/Taitec Inc.) with a cooled charge-coupled device camera and assembled in an Apple G4 computer using Adobe Photoshop 5.0. Signal intensity was quantified with a LumiVision Image analyzer. The percentages of DNA-bound EBNA1 in the presence ({circ}) or absence (•) of PAA were calculated from the signal intensity with the sum of the value for Triton X-100 soluble and insoluble EBNA1 as 100%.

 
An anti-BMRF1 protein-specific monoclonal antibody, clone name R3, was purchased from Chemicon Inc. An anti-PCNA mouse monoclonal antibody and Alexa 488- or Alexa 594-conjugated anti-BrdUrd mouse monoclonal antibodies were purchased from BD Transduction Laboratories and Molecular Probes, respectively. The secondary goat anti-rabbit or mouse IgG antibodies conjugated with Alexa 488 or 594 were purchased from Molecular Probes.

Biochemical Cellular Fractionation and Analysis of the Nuclear Associated Viral Proteins—Tet-BZLF1/B95-8 cells (1.5 x 107) treated with or without doxycycline at a concentration of 2 µg/ml were cultured and harvested at the indicated times. The cells were washed twice with phosphate-buffered saline at room temperature and lysed for 10 min on ice with 1 ml of ice-cold 0.5% TX100-mCSK buffer (10 mM Pipes, pH6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 0.5% Triton X-100) containing multiple protease inhibitors (25 µl/ml; protease inhibitor mixture for mammalian cell extracts, Sigma) 200 µM Na3VO4, and 20 mM NaF. The samples were then subjected to centrifugation (2000 x g for 3 min at 4 °C) to obtain Triton X-100-extractable fractions. The Triton X-100-extracted nuclei were digested with 250 units/ml DNase I (10 units/µl; Roche Molecular Biochemicals) in 0.1%TX100-mCSK containing 200 mM NaCl and 4 mM MgCl2 at 25 °C for 20 min. The samples were then centrifuged to obtain the solubilized chromatin fraction and the remaining non-chromatin nuclear structures. Each sample was adjusted to the same volumes by adding 2x SDS sample buffer and boiling, and aliquots corresponding to 1.8 x 104 cells per each lane was applied for SDS-PAGE.

EBV Genome-wide ChIP Assays—Genome-wide ChIP assays essentially as described (30) were modified as follows. Briefly, 5 x 107 Raji or B95-8 cells per sample were cultured in 100-mm plates in the presence or absence of 2 µg/ml doxycycline. After the cells were harvested and washed twice with PBS, cells were lysed in 0.5% TX100-mCSK buffer for 10 min on ice and centrifuged (2000 x g for 5 min) to obtain Triton X-100-extractable fractions. The Triton-extracted nuclei were cross-linked in situ with 5 ml of 1% formaldehyde in 0.5% TX100-mCSK buffer for 20 min at room temperature and then centrifuged at 2000 x g for 5 min. Cell pellets were suspended in solution I (5 M urea, 10 mM EDTA, and 0.2 M NaCl) at 4 °C and centrifuged, resuspended in solution I, and sonicated to reduce the average DNA fragment to 500–1000 bp in length (verified on agarose gels; data not shown). Cell extracts (1 ml) were then adjusted to 1.42 g/cm3 CsCl (567.8 mg/ml) brought to 5 ml with Tris-EDTA buffer, centrifuged in a Beckman SW55 Ti rotor at 40,000 rpm for 72 h, and 0.2 ml of the fractions were collected from the top. Eight fractions containing cross-linked protein-DNA were pooled and dialyzed overnight at 4 °C against a buffer containing 5% glycerol, 10 mM Tris-HCl, pH 8, 1 mM EDTA, and 0.5 mM EGTA using Slide-A-Lyzer cassettes (Pierce). After dialysis, samples were reacted for 1 h with 1.5 µg of the anti-EBNA1-specific antibody or 1.5 µg of non-immune rabbit IgG as control. Immune complexes of proteins cross-linked to DNA were precipitated with protein A-Sepharose CL4B beads. After antigen-DNA complexes were eluted from the protein A beads in Tris-EDTA buffer containing 0.5% SDS, they were treated with 0.7 mg/ml proteinase K for 5 h at 45 °C. Protein-DNA cross-links were reversed by incubation at 65 °C for 6 h, and then DNA was purified using a PCR purification kit from Qiagen.

ChIP probes were prepared by modified round A/B/C random amplification of DNA (30). This is a simpler method for sequence-independent DNA amplification to flank unknown sequences with known sequences to allow performance of PCR. Primer A (5'-GTTTCCCAGTCACGATCNNNNNNNNN-3') has a nine-nucleotide random 3'-segment and a 17-nucleotide 5'-portion of defined sequence. The DNA was denatured by a 2-min incubation at 97 °C and cooled to 4 °C to let 4 mM primer anneal to random sites. Annealed DNA was added to 5 ml of buffer A (40 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 50 mM NaCl, 300 µM each dNTP, 5 mM dithiothreitol, 50 µg/ml bovine serum albumin, and 1 unit of T7 DNA polymerase without 3' -> 5'-exonuclease activity (Sequenase Version 2.0, USB)). The temperature was ramped to 37 °C over an 8-min interval and then maintained there for 8 min, resulting in the synthesis of the first strand of DNA. The T7 DNA polymerase was chosen for this step because it functions well at the low temperatures at which the random priming complexes are stable and because it possesses strand displacement synthesis capability in the absence of the exonuclease activity. Strand displacement synthesis enables the enzyme to synthesize long stretches of DNA by displacing other primers that have already annealed to the DNA. After denaturation and annealing, the synthesis step with the T7 DNA polymerase was repeated once more by adding fresh enzyme in 2.5 µl of buffer. In this second synthesis step, primer A will also prime on the products from the first round. The products of this second synthesis step are now suitable for PCR amplification; an unknown stretch of genomic DNA is flanked 5' by the known sequence of the primer and 3' by the inverted complement of this sequence. After round A reactions, samples were diluted with water to a final volume 60 µl.

PCR was carried out about 20 cycles, depending on the amount of starting material, with 15 µl of round A template, PCR buffer, Taq DNA polymerase (Invitrogen), 2 mM each dNTP, and 1 µM primer B (5'-GTTTCCCAGTCACGATC-3'). The primer B sequence is identical to the 17 5'-nucleotides of primer A. Amplified PCR products were purified through a Chroma Spin-30 column and labeled with 32P by PCR labeling; PCR was carried out for five cycles with above buffer in the presence of [32P]TTP, 15 µl of purified DNA, and 1 µM primer B (5'-GTTTCCCAGTCACGATC-3'). A BamHI library of EBV genomic DNA (0.05 pmol of each molecule) was dot-blotted onto a Hybond-N membrane (Amersham Biosciences), and hybridizations were performed with the 32P-labeled ChIP probe. After washing and exposure to an Imaging Plate, the signal intensity was analyzed with a BAS2500 Image analyzer and Image Guider software (Fuji Film Co. Ltd). The raw data were corrected as follows. Hybridization signals from anti-EBNA1 ChIP samples were reduced by the amount of raw signal from the control sample of an equivalent number of cells (non-immune rabbit IgG ChIP). Radiolabeled DNA did not hybridize to pBR322 or pUC18 vector DNA.

Immunofluorescence Analysis—All staining procedures were carried out at room temperature except for extraction and incubation with primary antibodies. For immunofluorescence experiments, newly synthesized DNAs were labeled by incubating Tet-BZLF1/B95-8 cells with 10 µM BrdUrd added directly to the incubation medium for 1 h prior to harvesting. Cells were washed with ice-cold PBS and extracted with 0.5% TX100-mCSK buffer on ice for 2 min. Multiple protease inhibitors (25 µl/ml; Sigma), 200 µM Na3VO4, and 20 mM NaF were also added to the buffer. Cells were fixed with 70% methanol for 30 min on ice. The fixed cells were washed with PBS and permeabilized with 0.05% Triton X-100 in PBS for 15 min. The cells were blocked for 1 h in 10% fetal calf serum in PBS and then incubated for 1 h with each primary antibody diluted in PBS containing 10% fetal calf serum. The anti-PCNA mouse monoclonal antibodies and control IgG were used at 2.5 µg/ml. The anti-EBNA1 rabbit polyclonal antibody was used at 2.3 µg/ml, and control rabbit IgG was used at 5 µg/ml. The anti-BMRF1 mouse monoclonal antibody was used at 5 µg/ml. The samples were then incubated for 1 h with the secondary goat anti-rabbit or mouse IgG antibodies conjugated with Alexa 488 or 594. Neither of the control antibodies yielded specific signals in either single-staining or double-staining experiments.

For the staining of BrdUrd incorporated into DNA, cells were treated for 10 min with 2 N HCl containing 0.5% Triton X-100 to expose the incorporated BrdUrd residues before blocking. The cells were washed twice with PBS and neutralized with 0.1 M sodium tetraborate (pH 9) for a 5-min incubation. For the BrdUrd staining, an Alexa 488- or Alexa 594-conjugated anti-BrdUrd mouse monoclonal antibody was used at 5 µg/ml.

All washes after antibody incubation were carried out with PBS containing 0.1% Tween 20. The samples were mounted on Vectashield (Vector Laboratories), and image acquisition was performed using a Bio-Rad Radiance 2000 confocal laser-scanning microscope equipped with a PlainApo 100 x 1.4 NA oil-immersion objective lens. Images were processed and assembled in an Apple G4 computer using Adobe Photoshop 5.0.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Biochemical Fractionation Reveals Subcellular Localization of EBNA1 during the Latent Phase—We have previously revealed subcellular dynamics of human chromosomal DNA replication initiation proteins such as the origin recognition complex, CDC6, and the mini-chromosome maintenance (MCM) protein complex in HeLa cells using biochemical fractionation methods (29, 31). We applied this method to the Tet-BZLF1/B95-8 cells. In the method, cells are first treated with a buffer containing non-ionic detergent Triton X-100 in relatively physiological condition that extracts not only cytoplasmic but also nuclear proteins not tightly bound to nuclear structures. The Triton X-100 treatment disrupts nuclear envelopes but the nuclear lamina remains intact, thus maintaining nuclear structures (32). It was observed that cellular membranes disappeared, but nuclei remained intact morphologically as judged by microscopic analyses (data not shown). The proteins remaining in the extracted nuclei have been shown to indeed bind to the chromatin/nuclear matrix and thus represent functionally active fractions (29, 31). Second, the extracted nuclei were digested with DNase I to remove the bulk of the host and EBV genomes. With these procedures, about two-thirds of the MCM4 and MCM7 proteins were extracted with the buffer, and most of the remainder were solubilized after DNase I treatment, demonstrating their chromatin binding (Fig. 1). The core histones were detected only in the nuclear pellet fractions, and release of the nucleus-bound MCM4 and MCM7 by DNase I digestion was accompanied by the liberation of core histones. On the other hand, almost all of the CDC6 protein was detected in the detergent-unextracted fraction, but, in contrast with the chromatin-bound MCM proteins, the nucleus-bound CDC6 was resistant to DNase I treatment (Fig. 1). Thus, the CDC6 protein was in nuclear matrix-bound form. These data are consistent with the conclusion obtained previously from biochemical fractionation of HeLa cells (29) confirming the reproducibility of the fractionation procedure in Tet-BZLF1/B95-8 cells.



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  		FIG. 1.
Biochemical analysis of the subcellular distribution of CDC6, MCM4, MCM7, EBNA1, and histone proteins in Tet-BZLF1/B95-8 cells. Whole cell lysate (W) was prepared from asynchronous Tet-BZLF1/B95-8 cells without induction, whereby ~60–70% cells are in the G1 phase. Triton X-100-extractable supernatants (S) and extracted nuclear pellets (P) were prepared as described under "Experimental Procedures." The extracted nuclei were further treated with DNase I to obtain DNase I-solubilized supernatants containing chromatin and EBV genomes (S), and the remainder were insoluble non-chromatin nuclear pellets (P). These fractions were separated by SDS-PAGE and immunoblotted with anti-CDC6, anti-MCM4, anti-MCM7, and anti-EBNA1, respectively. The samples were also subjected to 15% SDS-PAGE followed by Coomassie Blue staining for the detection of core histones.

 
The same fractions were then examined with an anti-EBNA1 antibody (Fig. 1). About four-fifths of the EBNA1 was detected in the extractable nucleoplasmic fraction, and the rest of the nucleus-bound EBNA1 was as sensitive to DNase I treatment as were the chromatin-bound MCM proteins. Similar results were also obtained with Raji human Burkitt's cell line (data not shown), indicating that most EBNA1 exists as a DNA-unbound form and that part of the protein binds to DNA in the latent phase of EBV replication.

Although Total Levels of EBNA1 Are Constant, the Levels of DNA-bound Form of EBNA1 Are Increased as Lytic Infection Proceeds—To investigate whether the levels of EBNA1 change during the lytic replication, we prepared whole cell lysates from lytic program induced-Tet-BZLF1/B95-8 cells at the indicated times and analyzed them by immunoblotting. As shown in Fig. 2B, the levels of EBNA1 were unchanged among the samples. Thus, levels of EBNA1 are relatively constant throughout the productive infection. As shown in Fig. 2A, the levels of EBNA1 vary among cell lines. The expression level in the Tet-BZLF1/B95-8 cells was slightly lower than those in Akata, Raji, and parental B95-8 cells.

During the latent phase of the cells most EBNA1 was in detergent-soluble form, and only less than one-fifth of the proteins bound to DNA (Figs. 1 and 2C, row marked 0h). After the induction of lytic replication, the levels of the DNA-bound form were increased with time to attain almost half of the total after 48 h post induction (Fig. 2, C and D), although the actual total levels were relatively constant (Fig. 2B). The herpes virus DNA polymerase inhibitor phosphonoacetic acid (PAA) does not inhibit chromosomal DNA replication at all but prevents lytic viral DNA replication, although EBV immediate-early and early proteins are expressed (26). In the presence of PAA, the levels of the DNA-bound form of EBNA1 were almost constant before and after the addition of doxycycline (Fig. 2, C and D). These observations strongly suggest that EBNA1 actively binds to newly synthesized EBV genomes during lytic replication.

EBNA1 Binds to oriP Regions of the EBV Genome during EBV Latent Infection as Revealed by EBV Genome-wide ChIP Assay—We directly investigated the in vivo dynamics of EBNA1-oriP interaction by ChIP assay. To cross-link EBNA1 proteins at their sites of interaction with EBV genome DNA, we added formaldehyde to Raji or B95-8 cells after treatment with 0.5% TX100-mCSK buffer. The cells were then suspended in a buffer containing 5 M urea and sonicated to shear chromatin and EBV genomes to an average size of <1 kb, followed by separation of the cross-linked DNA by cesium chloride density gradient centrifugation. To isolate EBNA1-DNA complexes, immunoprecipitation reactions with anti-EBNA1 polyclonal antibody or non-immune control IgG were performed. In our approach, the immunoprecipitated DNA sequences were amplified by PCR, radiolabeled with 32P, and then used to probe dot blots of a series of plasmids encompassing almost the entire EBV genome (~170 kb; Fig. 3A). The results allowed us to identify which segments of the EBV genome were enriched in the immunoprecipitation and thereby make an EBV genome-wide map of the in vivo EBNA1-DNA interaction. To determine the specific genomic targets of EBNA1, we twice repeated immunoprecipitation with the anti-EBNA1 antibody independently, each time in parallel with a control immunoprecipitation using the same amounts of IgG against Raji and B95-8 cells. A single site of EBNA1-DNA interaction would allow immunoprecipitation of randomly sheared EBV genomic DNA fragments (0.5–1 kb) that hybridized not only to the DNA segment representing the actual binding site but also to its genomic neighbors.



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  		FIG. 3.
Mapping of EBNA1 binding sites by EBV genome-wide ChIP method. A, a BamHI restriction endonuclease map of the B95-8 EBV genome. The oriP region containing the FR and the DS are indicated by shaded rectangle boxes in the expanded map of the BamHI-C DNA fragment. The DNA fragments indicated at the bottom were cloned into the pUC18 vector. B, hybridization of an EBV genome BamHI library by the genome-wide ChIP method using anti-EBNA1 IgG or control IgG. Raji cells were subjected to ChIP analysis as described under "Experimental Procedures." Cross-linked genomic DNA was prepared and purified by CsCl2 centrifugation prior to immunoprecipitation using anti-EBNA1 rabbit IgG or non-immune rabbit IgG as a negative control. The isolated DNA was amplified by PCR using random primers, radiolabeled with 32P, and used to probe dot blots of the EBV BamHI DNA library plasmids shown (panel A) or the pBR322 vector in the presence of 300 µg/ml cold competitor, high complexity HeLa DNA. EBV genome-wide ChIP assays were performed as described under "Experimental Procedures." The top section illustrates the blotting pattern of cloned EBV genome BamHI DNA fragments spotted on the filter membrane. The middle and bottom sections show the images obtained after hybridization with radiolabeled PCR-amplified DNA fragments enriched by control IgG and anti-EBNA1 antibodies, respectively, as the probe. C, quantification of EBNA1 binding to the BamHI library of the EBV genome by genome-wide ChIP assays with the cross-linked Raji cell sample. Hybridization signals obtained by a bio-imaging analyzer system (BAS2500, Fuji Film Co. Ltd.) with control rabbit non-immune IgG ChIP samples were subtracted from the signals obtained with the anti-EBNA1 antibodies in the same imaging plate. Relative activities of quantitative autoradiographs were expressed as counts of photo-stimulated luminescence units, using the results quantified with Image Gauge software and a BAS2500 image analyzer (Fuji Film Co. Ltd.). The data were then adjusted for hybridization efficiencies to the 24 plasmids by normalizing to the hybridization signals obtained with radiolabeled total genomic DNA from HeLa cells. The corrected data are plotted ± S.D. and are obtained from duplicate dot blots. All blots can be compared with each other qualitatively. D, EBNA1 binds specifically to oriP regions within BamHI-C DNA fragments. Quantification of EBNA1 binding to DNA fragments was performed by ChIP assays with cross-linked Raji cell samples.

 
The results obtained, as shown in Fig. 3, B and C, clearly demonstrated that EBNA1 binds directly to a BamHI-C DNA fragment containing oriP and only slightly to BamHI-F and -Q fragments in Raji cells. Furthermore, as shown in Fig. 3D, fine mapping within the EBV BamHI-C fragment was carried out to allow detection of any differences in distribution between the oriP region and the flanking regions (see map in Fig. 3A). Preferential immunoprecipitation of oriP regions, including FR and DS by the anti-EBNA1 antibody, was clearly demonstrated. Equivalent experiments were performed with other EBV latently infected Daudi and B95-8 cells, and similar results were obtained (data not shown). It has been reported that EBNA1 binds to FR and DS elements in oriP regions in vitro and also to a downstream region of Q promoters to negatively regulate the transcription from F/Q promoters during latent III infection (33). Also, EBNA1 binding to the oriP in vivo has been postulated on the basis of dimethyl sulfate and ligation-mediated PCR footprinting analyses (9, 34). Our data give direct proof of EBNA1 binding to oriP in vivo. When cross-linking by formaldehyde was carried out before 0.5% TX100-mCSK treatment, the genome-wide ChIP assay more obviously detected BamHI-Q and -F fragments (data not shown) in addition to those of BamHI-C, suggesting weak binding of EBNA1 to the Q promoter region. We found no other high affinity region for EBNA1 binding within the entire EBV genome in Raji cells. In addition, to examine whether changes in the levels of EBNA1 binding to oriP are dependent on the cell cycle, we prepared asynchronous (where 60–70% cells are in G1 phase), early S phase (hydroxyurea-treated), and G2/M phase (nocodazole-treated) Raji cells and applied them to the ChIP assay. EBNA1 binding to oriP was constantly observed (data not shown), indicating EBNA1 binding to oriP throughout the cell cycle in agreement with previous reports (9, 34, 35). Taken together, these observations confirmed previous findings more directly and verified the accuracy of the EBV genome-wide mapping methods for a survey of EBNA1 binding sites.

EBNA1 Binds to the oriP Region during Lytic Replication, and Bound Levels Increase with Its Progression—Biochemical fractionation analyses revealed that the levels of DNA-bound EBNA1 were increased with time after the induction of lytic replication (Fig. 2C). We therefore focused on whether EBNA1 then binds to oriP regions of the EBV genome or dissociates to bind to other regions or chromatin DNA. As shown in Fig. 4, EBV genome-wide ChIP analyses revealed that EBNA1 still binds to the oriP region after the induction of lytic replication and that the levels of EBNA1 binding to regions containing FR or oriP become augmented as lytic replication progresses. In contrast, the signals for the BamHI-A DNA fragment (Fig. 4A) and for other BamHI restriction fragments (data not shown) were very low and unchanged throughout. Similar experiments were also performed in the presence of PAA to inhibit viral lytic replication (Fig. 4). In contrast to the case in the absence of PAA, the levels of EBNA1 binding to regions containing FR or oriP were almost unchanged before and after the addition of doxycycline. Thus, EBNA1 binds tightly to oriP not only before but also after the induction of lytic replication, and its augmentation is dependent on increased levels of viral DNA synthesized during lytic replication.



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  		FIG. 4.
Increasing levels of EBNA1 binding to oriP after the induction of lytic replication in Tet-BZLF1/B95-8 cells. Lytic replication was induced in Tet-BZLF1/B95-8 cells by the addition of 2 µg of doxycycline per milliliter in the presence ({circ}) or absence (•) of PAA (400 µg/ml), and cells were harvested at the indicated times thereafter and subjected to ChIP analysis as described under "Experimental Procedures." Cross-linked genomic DNA was isolated using anti-EBNA1 or non-immune rabbit IgG as a negative control. The isolated DNA was radiolabeled and used to probe dot blots of EBV BamHI library plasmids and the five plasmids containing DNA fragments within the BamHI-C fragment of the EBV genome (see Fig. 3A) or the pUC18 vector (0.05 pmol of each plasmids). Data for BamHI A (A), the plasmid KS containing only the FR region of oriP (B), the plasmid KORI containing both FR and DS (C), and the plasmid pBamC3 containing a DNA fragment neighboring the DS region (D) are presented. Hybridization signals obtained with control rabbit non-immune IgG were subtracted from the signals obtained with the anti-EBNA1 IgG. The average corrected data obtained from duplicate dot blots were plotted. All blots can be compared with each other qualitatively.

 
Intranuclear Localization of EBNA1 before the Induction of Lytic Replication—To further examine the subnuclear distribution of EBNA1 during latent infection, we utilized an immunostaining procedure. Accordingly, the Tet-BZLF1/B95-8 cells were co-stained using anti-EBNA1 and anti-PCNA or anti-BrdUrd antibodies after extraction with 0.5% TX100-mCSK buffer (Fig. 5). PCNA, a homotrimeric clamp at replication forks that has been well studied by microscopic observations, is a widely used marker for cellular replication foci (36). Thus, PCNA foci appear at S phase in cell cycles. When Tet-BZLF1/B95-8 cells were stained with anti-PCNA antibodies before induction, fine focal staining of PCNA was clearly observed in S phase nuclei (Fig. 5B). However, PCNA foci were not apparent in most of the cells, as shown in Fig. 5A, because they were asynchronous (>60% in G1 phase). In contrast, it was found that EBNA1 proteins were distributed diffusely in nuclei throughout the cell cycle with a fine granular pattern (Fig. 5), in line with the diffuse punctate localization in the nuclei of interphase cells reported earlier (37). As shown in Fig. 5B, it should be noted that the EBNA1 foci hardly overlapped with the PCNA replication foci in EBV latently infected cells. In addition, BrdUrd staining in the majority of the S phase cells revealed a fine granular pattern of DNA replication sites, indicating that cellular DNA replication occurs at numerous discrete sites in the nucleus, again with only a partial overlap with EBNA1-staining (Fig. 5C).



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  		FIG. 5.
Intranuclear distribution patterns of EBNA1, PCNA and newly synthesized DNA during latent infection. Tet-BZLF1/B95-8 cells were cultured in the absence of doxycycline. Newly synthesized DNA was labeled by incubating with 10 µM BrdUrd for 1 h prior to harvesting and treatment with 0.5% TX100-mCSK buffer. The nonionic detergent-extracted cells were fixed with methanol and processed as described under "Experimental Procedures." The immunostained cells were processed for confocal microscope analyses. Panels A and B shows profiles of EBNA1 and PCNA immunostained using the anti-EBNA1 rabbit polyclonal antibody followed by the Alexa 488-conjugated anti-rabbit IgG second antibody and the anti-PCNA mouse monoclonal antibody followed by the Alexa 594-conjugated anti-mouse IgG second antibody. Panel C shows profiles of EBNA1 and BrdUrd (BrdU) incorporated newly synthesized DNA immunostained using the anti-EBNA1 rabbit polyclonal antibody followed by the Alexa 488-conjugated anti-rabbit IgG second antibody and the Alexa 594-conjugated anti-BrdUrd monoclonal antibody. Right column contains merged images.

 
Using confocal microscopy analyses, Ito and Yanagi showed EBNA1 proteins to be highly colocalized with PCNA or RP-A foci representing the replication foci of chromatin DNA during S phase in the Chinese hamster ovary-derived epithelium-like cell line CHO-K1 in the absence of oriP plasmids or the EBV genome (38). They suggested that EBNA1 is associated not only with cellular metaphase chromosomes but also with condensing chromatin/chromosomes and probably with interphase chromatin, especially with its newly replicated regions. However, we could scarcely detect any colocalized images of EBNA1 and PCNA foci in EBV latently infected B95-8 cells (Fig. 5). In the absence of the EBV genome, EBNA1 might behave differently, interacting with chromatin DNA directly or indirectly mediated by cellular proteins.

Redistribution of EBNA1 and Its Positional Relationship to the Replication Compartments during Lytic Replication—The biochemical and ChIP analyses strongly indicate that EBNA1 binds to oriP regions of a newly replicated EBV genome during lytic replication. We therefore investigated localization of EBNA1 in relation to lytic replication compartments by an immunostaining procedure (Fig. 6). To examine whether EBNA1 co-distributes with BrdUrd-substituted newly synthesized DNA sites, cells were double stained with anti-EBNA1 and anti-BrdUrd antibodies after extraction with 0.5% TX100-mCSK buffer. Interestingly, the numerous fine granular spots of EBNA1 seen in the latent phase (Fig. 5) appeared to gather and redistribute to the sites of viral DNA synthesis, replication compartments (Fig. 6A). Thus, EBNA1 proteins were found as clear granular dots within or juxtaposed to the replication compartments where BrdUrd was incorporated (Fig. 6, A and B, bottom rows). The number of EBNA1 foci was decreased compared with those in the latent phase, but their sizes were much increased. At the early stage of lytic replication, the cohesion of EBNA1 proteins might take place in replication compartments. At late stages the number of EBNA1 proteins was increased, and EBNA1 foci were observed as multiple dots in the replication compartments. The images were similar to those of the double staining of EBNA1 and the BMRF1 Pol processivity factor (Fig. 6B), because the staining patterns of BrdUrd-incorporated DNA and BMRF1 proteins coincided completely (data not shown). We observed the redistribution of EBNA1 within viral replication compartments in other clones of lytic replication-induced Tet-B95-8 cells or Burkitt's human Akata cell line (data not shown). Thus, BMRF1 proteins appear to be widely distributed on newly synthesized DNA and cover the EBV genome entirely, whereas EBNA1 binds tightly to only oriP regions of the EBV genome. Taken together, these data further demonstrate EBNA1 binding to the oriP region on newly synthesized EBV genomes.



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  		FIG. 6.
Redistribution of EBNA1 to viral replication compartments after the induction of lytic replication assessed by immunostaining and confocal microscopic analyses. Tet-BZLF1/B95-8 cells were cultured in the presence of 2 µg of doxycycline per milliliter, and newly synthesized DNA was pulse-labeled by incubation with 10 µM BrdUrd (BrdU) for 1 h prior to harvesting and treatment with 0.5% TX100-mCSK buffer. The nonionic detergent extracted cells were fixed with methanol and processed as described under "Experimental Procedures," followed by application to confocal laser microscopy. Panel A shows profiles of EBNA1 and BrdUrd-incorporated newly synthesized DNA immunostained using the anti-EBNA1 rabbit polyclonal antibody followed by the Alexa 594-conjugated anti-rabbit IgG second antibody and the Alexa-488-conjugated anti-BrdUrd monoclonal antibody. Panel B shows profiles of EBNA1 and the BMRF1 protein immunostained using the anti-EBNA1 rabbit polyclonal antibody followed by the Alexa 488-conjugated anti-rabbit IgG second antibody and the anti-BMRF1 mouse monoclonal antibody followed by the Alexa 594-conjugated antimouse IgG second antibody. Second column from the right shows merged images. Magnified images of individual replication compartments are shown in the far right column.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using the footprinting method, EBNA1 binding to four binding sites within DS in vitro was shown (10, 15). The same conclusion was drawn from studies of in vivo footprinting on oriP in Raji cells throughout the cell cycle (9, 34). It has been reported, however, that cellular proteins of sizes 40 and 60 kDa that could bind to the EBNA1 binding site of the 30-bp repeats of oriP, as revealed by DNase I footprinting, can be induced in an EBV-negative Burkitt's lymphoma cell line by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (39). It is possible that these cellular proteins would give dimethyl sulfate footprints indistinguishable from those of EBNA1 or would distort DNA structure at the oriP in the manner of EBNA1. Thus, the footprinting methods have not provided direct evidence of EBNA1 binding to oriP in vivo. Quantitating protein-DNA interactions in vivo has proven much more difficult than in vitro, and, thus, binding data are in fact limited for most classes of protein. Over the past ten years, however, methods for covalently cross-linking proteins to DNA in vivo have been developed that can measure the interaction of sequence-specific DNA-binding proteins with genomic DNA. In several other studies, DNA obtained in ChIP assays can be analyzed by polymerase chain reaction with primer sets that recognize relatively circumscribed region(s) of interest (e.g. promoters or discrete origins of replication). With regard to the oriP region, it has been thus demonstrated recently that this region binds to EBNA1 (18, 40, 41). However, given the large size and more complex nature of the entire EBV genome, it was impossible to predict a priori how the proteins might be distributed in the genome directly. We therefore opted for a more unbiased approach in which we could determine the distribution of EBNA1 proteins throughout the 170-kb region encompassing the whole EBV genome. The present EBV genome-wide survey of DNA binding by EBNA1 in vivo is the first such report, to our knowledge, confirming that EBNA1 only binds tightly to the oriP region.

EBV genome-wide ChIP analyses revealed that EBNA1 still binds to the oriP region after the induction of lytic replication and that the levels of EBNA1 binding to regions containing FR or oriP become augmented as lytic replication progresses. Experiments in the presence of the viral DNA polymerase inhibitor PAA clearly confirmed that the binding of EBNA1 to the oriP region is dependent on increased levels of viral DNA synthesized during lytic replication. It should be noted that we have not shown directly the binding of EBNA1 to oriP during lytic infection to be dependent on the binding site of EBNA1.

In our previous studies, chromatin was prepared by isolating a nuclear pellet from cytoplasmic and soluble fractions in the presence of Triton X-100 or an equivalent detergent (29, 31). Such detergents are known to disrupt the nuclear envelope and, in addition, lead to solubilization and extraction of nuclear proteins (32). The solubilized proteins fractionate with the cytoplasmic supernatant, whereas the insoluble, detergent-resistant proteins fractionate with the nuclear pellet. In our ChIP assay, anti-EBNA1 immunoprecipitation enriched mainly the oriP regions and only slightly the BamHI-F and Q fragments, because formaldehyde fixation was carried out after 0.5% TX100-mCSK treatment (Fig. 3 and 4). When cross-linking by formaldehyde was performed before the detergent extraction, the genome-wide ChIP assay also clearly detected the BamHI-Q and -F fragments as well as the BamHI-C fragment (data not shown), although direct fixation of cultured cells with formaldehyde elevates nonspecific protein-protein or protein-DNA interactions in proportion to the reaction time. In light of this difference, our data suggest weak binding of EBNA1 to the Q promoter region in vivo. To clarify the sites of tight EBNA1-EBV genome interaction, we employed formaldehyde fixation carried out after detergent treatment.

Given the oriP binding property of EBNA1 and the colocalization of EBNA1 with oriP regions in the form of dots in the nuclei, the number of EBNA1 foci in EBV-latently infected cells should reflect the copy number for EBV genomes. We observed numerous EBNA1 foci with dozens of distinct dots and many other faint spots in Tet-BZLF1/B95-8 cells before induction. Because EBNA1 binds not only to oriP regions but also to cellular chromosomes through all phases of the cell cycle, including mitosis (20, 42) and interphase (42–44), most of the faint punctate spots might reflect the latter.

The present study demonstrated that EBNA1 binds to oriP regions of the EBV genome not only during latent infection but also the viral lytic cycle. The sites of EBNA1 localization in the replication compartments are found as discrete distinct spots. Although the number of these spots was obviously lower than in latent phase (compare Fig. 5 with Fig. 6), the size was clearly increased. In fact, oriLyt-mediated DNA replication is thought to be biphasic; an early {theta}-like mode is followed by a complex pattern that could result from rolling circle DNA replication. Pfuller and Hammerschmidt demonstrated that oriLyt replicated semiconservatively soon after induction of the lytic cycle and that oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA, which has far fewer negative supercoils apart from the multimeric forms and high molecular weight DNA (45). We speculate that oriP regions of template genomes and/or newly synthesized EBV monomeric plasmid genomes at early stage of lytic replication cluster to the spots mediated by EBNA1 proteins.

The oriP region is reported to be attached to the nuclear matrix during the latent phase (46, 47). Our present data, however, revealed that EBNA1 is easily solubilized by nuclease treatment, suggesting that its affinity for the nuclear matrix appears to be not so high. In a previous study, a nuclear matrix was prepared by classical high salt extraction after nuclease digestion (46, 47). However, it has been pointed out that this procedure can lead to an artificial result due to non-physiological buffer conditions. In our procedure, nuclease digestion and subsequent chromatin elution are performed under relatively physiological conditions. These different procedures could be one possible reason for the seeming discrepancy. Although it is clear that oriP can function as an efficient replication origin on short plasmid (13), initiation frequently occurs from other regions rather than from oriP on the viral genome (48). The issue of whether oriP binds to nuclear matrix under physiological condition should be addressed in future.

Our data strongly suggest that EBNA1 proteins, together with viral replication proteins, interact with newly synthesized viral DNA genomes during lytic replication, because these viral proteins were found to colocalize within viral replication compartments. The induction of a lytic program in Tet-BZLF1/B95-8 cells with doxycycline produces progeny viruses, thus completing the productive replication cycle. To examine whether these viral encoded proteins constitute portions of virus particles, we have purified EBV virions from the culture media of lytic program-induced Tet-BZLF1/B95-8 cells treated with doxycycline for 5 days. The purified virus particles were applied to sucrose density gradient centrifugation analyses, and the peak fractions containing virus particles were examined for the presence of the individual proteins by Western blotting. We could not detect EBNA1, BMRF1 Pol accessory factor, and BALF2 single-stranded DNA-binding protein in these fractions at all (data not shown), indicating that EBNA1 and viral replication proteins may dissociate from viral genome when the DNAs are packaged into viral capsids in nuclei.

The level of EBNA1 in Tet-BZLF1/B95-8 cells during latent infection were slightly lower than those in Akata, Raji, and B95-8 cells (Fig. 2A). Total levels of EBNA1 were almost constant before and after the induction of lytic replication, and less than one-fifth of EBNA1 proteins were in the DNA-bound form during latent infection. Although viral genomes were amplified >100-fold after the induction of lytic infection (26, 49), more than half of EBNA1 were in the DNA-bound form 72 h post induction (Fig. 2D). Thus, there should not be sufficient levels of EBNA1 to associate with replicating DNA. EBNA1 interacts not only with a viral genome but also with chromosomal DNA during latent infection (19–21). After the induction of the lytic phase, EBNA1 is redistributed to viral replication compartments. Therefore, it is possible that EBNA1 proteins binding to chromosomal DNA dissociate from chromosomal DNA and become associated with newly synthesized viral DNA. Alternatively, EBNA1 might not bind to all of the oriP regions on newly synthesized viral DNAs during lytic infection. Also, because EBNA1 is released from viral genomes by an unknown mechanism when packaging into viral capsids, recycling of EBNA1 during lytic infection might occur.


   FOOTNOTES
 
* This work was supported by Grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, Culture, and Technology of Japan 16017322 and 15390153 (to T. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both of these authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Tel.: 81-52-764-2979; Fax: 81-52-764-2979; E-mail: ttsurumi{at}aichi-cc.jp.

1 The abbreviations used are: EBV, Epstein-Barr virus; ChIP, chromatin immunoprecipitation; DS, dyad symmetry; EBNA1, EBV nuclear antigen 1; FR, family of repeats; MCM, mini-chromosome maintenance; mCSK buffer, modified cytoskelton buffer; PAA, phosphonoacetic acid; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; Pipes, 1,4-piperazinediethanesulfonic acid. Back

2 T. Daikoku, A. Kudoh, M. Fujita, Y. Sugaya, H. Isomura, N. Shirata, and T. Tsurumi, J. Virol., in press. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Shirakata (Tokyo Medical and Dental University) for oriP plasmids, KORI, and KS. We also thank Dr. Shirahige (RIKEN of Japan) for suggestions for the genome-wide ChIP assay.



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 ABSTRACT
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