Architecture of Replication Compartments Formed du
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病菌学杂志 2005年第6期
Division of Virology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya
Virology Division, National Cancer Center Research Institute, Chuohku, Tokyo, Japan
ABSTRACT
Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.
INTRODUCTION
Epstein-Barr virus (EBV) is a human herpesvirus that infects 90% of individuals. Primary EBV infection targets resting B lymphocytes, inducing continuous proliferation. In B-lymphoblastoid cell lines, only limited numbers of viral genes are usually expressed and there is no production of virus particles; this is called latent infection. In the latent state, EBV maintains its 170-kb genome as complete, multiple copies of plasmids. Latent-phase viral replication appears to faithfully mimic cellular replicons: EBV genomes or small oriP/EBNA-1 plasmids are synthesized only once in each S phase by the host cell replication machinery, following the rules of chromosome replication (28).
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 (7) and leads to an ordered cascade of viral gene expression: activation of early gene expression followed by the lytic cascade of viral genome replication and late gene expression.
Lytic replication differs from the latent amplification state in that multiple rounds of replication are initiated within oriLyt, and the replication process has a greater dependence on EBV-encoded replication proteins (12). In the viral productive cycle, the EBV genome is amplified more than 100-fold. Intermediates of viral DNA replication are found as large head-to-tail concatemeric molecules, probably resulting from rolling-circle DNA replication, which are subsequently cleaved into unit-length genomes and packaged into virions in the nucleus. The lytic phase of EBV DNA replication is dependent on seven viral replication proteins: the BZLF1 protein, an oriLyt binding protein; the BALF5 protein, a DNA polymerase (Pol); the BMRF1 protein, a Pol processivity factor; the BALF2 protein, a single-stranded-DNA binding protein; and the BBLF4, BSLF1, and BBLF2/3 proteins, which are predicted to be helicase, primase, and helicase-primase-associated proteins, respectively (6). It has been suggested that all except the BZLF1 protein conceivably work together at replication forks to synthesize leading and lagging strands of the concatemeric EBV genome (22).
It is generally accepted that nucleic acid metabolism, such as DNA replication and transcription, is carried out on spatiotemporally organized domain structures in the cell nucleus (16). Nonchromatin nuclear structures such as the nuclear matrix, the scaffold, and the nucleoskeleton have been suggested as key players in organizing high-order chromatin and nuclear structures (2, 3). In the case of DNA replication, for example, fluorescence microscopic analyses have revealed discrete granular sites of replication, i.e., replication sites or replication foci (17, 18). Replication foci may be constructed based on nonchromatin nuclear structures, since nascent DNA and many proteins involved in DNA synthesis have been found to attach to these (3, 13, 14). In the case of EBV lytic replication, it was previously demonstrated that the BZLF1 and BMRF1 proteins distribute diffusely in nuclei at the immediate-early stage and then redistribute and colocalize to common globular regions, called replication compartments, in the nuclei (20). Furthermore, it has recently been reported that upon lytic activation, interchromosomally located nuclear domain 10 becomes dispersed in the cells, and replicating EBV genomes were frequently found beside the nuclear domain (1). However, detailed analyses of the architecture of the replication compartments remain to be carried out.
We have previously established a biochemical fractionation method which enables us to detect the active fraction of cellular DNA replication initiation proteins that bind tightly to chromatin and nuclear matrix (10). Using this method, we have been studying the nuclear organization of the chromosomal initiation proteins and their spatiotemporal regulation (9, 10). In this study, taking advantage of this method and confocal microscopy analyses, we performed detailed and comprehensive analyses of the architecture of the replication compartments formed during EBV lytic replication.
MATERIALS AND METHODS
Cell culture. Tet-BZLF1/B95-8 cells (15) were maintained in RPMI medium supplemented with 1 μg of puromycin/ml, 250 μg of hygromycin B/ml, and 10% tetracycline-free fetal calf serum (FCS) (Clontech, Inc.). To induce lytic EBV replication, the tetracycline derivative doxycycline was added to the culture medium at a final concentration of 2 μg/ml. B95-8 cells were grown at 37°C in a 5% CO2 atmosphere in RPMI 1640 medium with 10% FCS. For induction of lytic replication in B95-8 cells, phorbol 12-myristate 13-acetate (TPA) at a final concentration of 100 ng/ml and 5 mM sodium n-butyrate were added to the culture medium. When blocking lytic DNA replication, phosphonoacetic acid (PAA), a herpesvirus DNA polymerase-specific inhibitor, was added to the culture medium at a final concentration of 400 μg/ml.
Antibodies. The anti-BRLF1 protein specific rabbit polyclonal antibody was prepared as follows. DNA fragments carrying the BRLF1 gene was amplified by PCR methods with a pair of primers, BRLF1-UP (5'-GGGAATTCGGGCATTTCCTCTGTTACTACTAGCCA-3') and BRLF1-DN (5'-TTAGCAATGCCTGTGGCTCATGCATAGTTT-3'). The DNA fragment containing a full-length copy of the BRLF1 gene was ligated between the EcoRI and HindIII sites downstream of the polyhedrin promoter region in pFastBac DUAL (Life Technologies). The method for the preparation of recombinant baculovirus was described previously (29). The BRLF1 protein was purified from Sf21 cells infected with the recombinant baculovirus AcBRLF1. The BRLF1 protein-specific rabbit polyclonal antibody was produced against the purified protein and was affinity purified with BRLF1 protein-coupled Sepharose 4B as described previously (10). The preparation and specificity of the anti-BZLF1 and -BALF2 rabbit polyclonal antibodies were described previously (26, 29). Anti-BALF5 specific rabbit antibody (25) was affinity purified with BALF5 protein-coupled Sepharose 4B as described previously (10). For double immunostaining with rabbit polyclonal antibodies, Alexa Fluor 594-conjugated anti-BALF5 protein specific rabbit antibody was prepared with Fluor dye materials, using the Alexa Fluor 594 protein labeling kit (Molecular Probes). After coupling, the Alexa Fluor 594-conjugated antibody was separated from free dye by gel filtration with Bio-Gel P-30 (Bio-Rad). Anti-BBLF2/3 specific rabbit antibody (29) was affinity purified with BBLF2/3 protein-coupled Sepharose 4B as described previously (10). An anti-BMRF1 specific monoclonal antibody, R3, and an anti-BRLF1 specific mouse monoclonal antibody were purchased from Chemicon and Argen Inc., respectively. The BZLF1 protein-specific mouse monoclonal antibody was purchased from Dako Inc. Alexa Fluor 488-conjugated antibromodeoxyuridine (anti-BrdU) mouse monoclonal antibody and secondary goat anti-rabbit or -mouse immunoglobulin G (IgG) antibodies conjugated with Alexa Fluor 488 or 594 were purchased from Molecular Probes.
Biochemical cellular fractionation and analysis of nucleus-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 were lysed for 10 min on ice with 1 ml of ice-cold 0.5%TX100-mCSK buffer [10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (pH 6.8), 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg of aprotinin per ml, 0.5% Triton X-100] containing multiple protease inhibitors (protease inhibitor mixture for mammalian cell extracts [Sigma], 25 μl/ml), 200 mM Na3VO4, and 20 mM NaF. The samples were then subjected to centrifugation (2,000 x g, 3 min, 4°C) to obtain Triton X-100-extractable fractions. The detergent-extracted nuclei were then digested with 250 U of DNase I (10 U/μl; Roche Molecular Biochemicals) per ml in 0.1%TX100-mCSK containing 2 mM MgCl2 at 25°C for 20 min. The samples were then centrifuged to obtain the solubilized chromatin fraction and the remaining nonchromatin nuclear structures. Each sample was adjusted to the same volume by adding 2x sodium dodecyl sulfate (SDS) sample buffer and boiled, and aliquots corresponding to 1.8 x 104 cells per lane was subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride membranes, and the proteins were detected with the enhanced chemiluminescence detection system (Amersham) as described previously (15). Images were processed by LumiVision PRO (Aisin/Taitec Inc.) with a cooled charge-coupled-device camera and assembled in an Apple G4 computer with Adobe Photoshop 5.0. Signal intensity was quantified with a LumiVision image analyzer.
Immunofluorescence analysis. All staining procedures except extraction were carried out at room temperature. For immunofluorescence experiments, newly synthesized DNAs were labeled by incubating Tet-BZLF1/B95-8 cells with 10 μM BrdU, 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 (Sigma; 25 μl/ml), 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% FCS in PBS and then incubated for 1 h with each primary antibody diluted in PBS containing 10% FCS or overnight with anti-BRLF1 rabbit polyclonal antibody at 4°C. The anti-BALF2, anti-BRLF1, anti-BZLF1, anti-BALF5, and anti-BBLF2/3 protein rabbit polyclonal antibodies were used at 5 μg/ml. The anti-BMRF1, anti-BZLF1, and anti-BRLF1 mouse monoclonal antibodies and the control IgG were also 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 Fluor 488 or 594. Neither of the control antibodies yielded specific signals in either single- or double-staining experiments.
For staining of BrdU-incorporated DNA, the cells were treated for 10 min with 2 N HCl containing 0.5% Triton X-100 to expose the incorporated BrdU residues before blocking. The cells were washed twice with PBS and neutralized with 0.1 M sodium tetraborate (pH 9.0) for 5 min. For the BrdU staining, Alexa Fluor 488-conjugated anti-BrdU 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 with a Bio-Rad Radiance 2000 confocal laser-scanning microscope equipped with a PlainApo 100x 1.4-numerical-aperture oil immersion objective lens. Images were processed and assembled in an Apple G4 computer with Adobe Photoshop 5.0.
Fluorescence in situ hybridization (FISH). The EBV BamHI-W fragment was labeled with Chroma Tide Alexa Fluor 594-5-dUTP (Molecular Probes, Inc.) by random primer labeling according to the procedure provided by the manufacturer and was used for the detection of amplified EBV genomes during lytic infection. The fragment was small enough to prevent the visualization of single episomes, thus enabling us to observe only EBV DNA that replicates upon induction of the lytic cycle, not latent EBV genomes.
Cells were fixed in 4% paraformaldehyde (PFA) in PBS (10 min at room temperature) or, when FISH was combined with immunocytochemistry, first immunostained as described above and then refixed in 4% PFA to cross-link bound antibodies. After being washed in PBS and permeabilized in 0.2% Triton X-100 (20 min on ice), cells were digested with RNase A (Qiagen) (100 μg/ml in PBS for 30 min at 37°C). The cells were then dehydrated in an ethanol series (70, 80, and 100% for 3 min each at 20°C), air dried, and immediately covered with a probe mixture containing 50% formamide in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), probe DNA (10 ng/μl), 10% dextran sulfate, salmon sperm DNA (0.1 μg/μl; Sigma), and yeast tRNA (1 μg/μl; Sigma). The probe and cells were simultaneously heated at 94°C for 4 min to denature DNA and incubated overnight at 37°C. After hybridization, specimens were washed at 37°C with 50% formamide in 2x SSC (twice for 15 min each) and 2x SSC (10 min). Finally, the cells were equilibrated in PBS and mounted in Vectashield (Vector Laboratories, Inc).
RESULTS
In a recent study we isolated latently EBV-infected Tet-BZLF1/B95-8 cells in which the exogenous BZLF1 protein is conditionally expressed under the control of a tetracycline-regulated promoter (15). As judged by flow cytometry analysis, more than 80% of Tet-BZLF1/B95-8 cells became positive for BZLF1 and BMRF1 proteins 24 h after doxycycline addition (15). The copy number of the viral genome in the cells was significantly amplified after 24 h postinduction (p.i.). After induction, the growth of the cells is completely arrested. Thus, the system is simple and highly efficient for conditional induction of the lytic replication program in the absence of any other external stimuli, allowing us to analyze precisely the architecture of the replication compartments of EBV lytic replication.
Biochemical fractionation to determine the subcellular localizations of EBV replication proteins during lytic infection. We have previously determined the subcellular localizations of mammalian chromosomal DNA replication initiation proteins such as origin recognition complex (Orc), CDC6, and minichromosome maintenance protein complex (MCM) in HeLa cells by using biochemical fractionation methods (9, 10). These proteins assemble sequentially to form prereplication chromatin. In this method, HeLa cells are first treated with a buffer containing the nonionic detergent Triton X-100 in relatively physiological conditions, which extract not only cytoplasmic proteins but also nuclear proteins that are not tightly bound to nuclear structures. The proteins remaining in the extracted nuclei have indeed been shown to bind to chromatin and nuclear matrix and thus represent functionally active fractions. Based on the findings obtained by this and other methods, we have suggested the spatiotemporal regulation of these initiation proteins (9, 10). For example, it has been demonstrated that human MCM2 to -7 proteins bind to chromatin as heterohexameric forms in G1 phase and dissociate as replication proceeds (11). In this study, we applied this method for analyzing the behavior of EBV replication proteins.
We first examined whether the method could be applied to analyses of Tet-BZLF1/B95-8 B-lymphoblastoid cells. Accordingly, Tet-BZLF1/B95-8 cells were treated with 0.5%TX100-mCSK buffer, and then the extracted nuclei were digested with DNase I to remove the bulk of chromatin and EBV genome. As control markers for subcellular localization, we employed the CDC6, MCM4, and MCM7 proteins, which are known to be components of the prereplication complex of chromosomal DNA replication. As shown in Fig. 1, about two-thirds of the MCM4 and MCM7 proteins were extracted with the buffer, and most of the remainder was solubilized after DNase I treatment, demonstrating their chromatin binding. On the other hand, almost all of the CDC6 protein was detected in the non-detergent-extractable fraction, but in contrast to the chromatin-bound MCM proteins, the nucleus-bound CDC6 was resistant to DNase I treatment (Fig. 1). Thus, the CDC6 protein was in a nuclear matrix-bound form. These data are consistent with those previously obtained with HeLa cells (9, 10), confirming the reproducibility of the fractionation procedure with Tet-BZLF1/B95-8 cells. It should be noted that sufficient amounts of multiple protease inhibitors should be added to the extraction buffer in order to inhibit proteolysis as much as possible.
To investigate the subcellular distribution of EBV-encoded replication proteins after induction of the lytic program, lytic replication was induced in Tet-BZLF1/B95-8 cells with doxycycline. Cells were harvested at the indicated times, and biochemical fractionation was performed. The kinetics of expression profiles of EBV replication proteins have been previously examined by immunoblot analysis (15). Before induction, expression of the lytic viral replication proteins was almost undetectable (Fig. 2). The BZLF1 protein became detectable at 4 h p.i. (data not shown) and reached a plateau at 48 h p.i. The other immediate-early protein, the BRLF1 protein, also appeared at 6 h p.i. with a plateau at 48 h p.i. Both the BZLF1 and BRLF1 proteins were strongly expressed at 24 h p.i. and continuously expressed to 72 h p.i. Biochemical fractionation analyses, as shown in Fig. 2, revealed that all the BZLF1 protein was detected in the non-detergent-extracted fraction and that the levels of the DNA-bound form, solubilized by DNase I treatment, increased with the progression of lytic replication. Like the BZLF1 protein, most of the BRLF1 proteins tightly bound to nuclei, and half of them were released by DNase I throughout lytic infection. These observations suggest that the BZLF1 and BRLF1 immediate-early proteins may be associated with the EBV genome throughout productive replication. Although from only these data we could not exclude the possibility that they also bind to cellular DNA, further immunostaining data indicated that this is not the case (see below).
The BALF2 single-stranded-DNA binding protein, the BALF5 polymerase catalytic protein, and the BMRF1 Pol accessory protein appeared after 12 h p.i. (data not shown) and reached a plateau at 24 h p.i. In contrast to the BZLF1 and BRLF1 proteins, about half of the BALF2 single-stranded-DNA binding protein and BALF5 Pol catalytic protein was extracted with 0.5%TX100-mCSK buffer at 24 h p.i. These fractions could be the excess proteins that do not function in situ. Most of the remainder was solubilized after DNase I treatment at 24 h p.i. (Fig. 2). The levels of the DNA-bound forms of both proteins increased as lytic replication proceeded (Fig. 2, 48 and 72 h). These fractions may be associated with replication forks on the replicated EBV genome. This notion was further supported by immunostaining data as described below.
As was reported previously (23), extremely large amounts of the BMRF1 proteins were found to be expressed in the lytic program-induced cells compared with the BALF5 Pol catalytic proteins as judged by Coomassie blue staining (data not shown). Almost all of the BMRF1 protein existed as a non-detergent-extractable DNA-bound form (Fig. 2), suggesting that the BMRF1 proteins might be widely associated with the EBV genome besides acting at viral replication forks as polymerase processivity factors. Again, this notion was supported by immunostaining data (see below).
The BMRF1 protein is localized in intranuclear focal sites of newly synthesized viral DNAs during lytic replication. Switching from the latent phase to viral productive replication causes complete arrest of cell cycle progression (15). Therefore, labeling with the thymidine analog BrdU and subsequent immunofluorescence analyses with confocal microscopy can be used to identify sites of viral DNA replication in B95-8 cells during lytic infections. Accordingly, the lytic program-induced Tet-BZLF1/B95-8 cells were labeled with BrdU for 1 h prior to harvesting and extracted with 0.5%TX100-mCSK buffer. It should be noted that this treatment strips non-DNA-bound form of viral or cellular proteins, making it useful to investigate the localization of functional fractions of viral replication proteins, as shown in Fig. 3. The cells were then fixed with methanol and stained with anti-BrdU rabbit antibody and anti-BMRF1 mouse monoclonal antibody after treatment with HCl for 10 min. The acid treatment little affected the distribution of BMRF1 and other proteins. We tested for specificity of the second antibodies and for reliability of discrimination with fluorescence microscopy filters. When cells were stained singly for either antigen with inappropriate combinations of first and second antibodies, no fluorescence was observed. Also, no immunofluorescence was observed with alternate filter.
BrdU staining appeared as small foci in the nuclei at early stages of viral DNA replication (Fig. 3A, upper panels). The staining pattern of BrdU, representing newly replicated DNA, completely coincided with that of the BMRF1 Pol accessory protein (Fig. 3A). Such globular structures were identified as the sites of EBV DNA localized in nuclei by FISH with a viral DNA probe (Fig. 3B), confirming the previous observations reported by Takagi et al. (20). Under the conditions used for FISH, we could not detect any signal of latently replicated viral genomes (data not shown). These sites of viral DNA synthesis have been termed replication compartments. The numbers of the spots representing replication compartments at early stages of the lytic replication were more than 10, which is lower than the average copy number (40 to 50 copies per cell) of the latent virus genome (Fig. 3A, upper panels). It is likely that only some of the latent viral genomes may become templates for lytic replication. With progression of lytic replication, the replication compartments became larger and appeared to fuse to form large globular structures that eventually filled the nucleus at late stages (Fig. 3), consistent with observations with herpes simplex virus type 1 (4, 21). As described above, the staining pattern of the BMRF1 Pol accessory protein completely coincided with the BrdU staining and localization of EBV DNA in the replication compartments (Fig. 3). Therefore, although the BMRF1 protein possesses an intrinsic double-stranded-DNA binding activity (23), it may bind to viral DNA preferentially and not to cellular chromosomal DNA. Furthermore, in the replication compartments, the BMRF1 proteins were distributed diffusely and homogenously, rather than as fine dots as was the case for the other viral replication proteins (see below). Thus, the immunostaining data together with the findings that almost all of the abundantly expressed BMRF1 protein molecules bound to DNA (Fig. 2A) indicate that BMRF1 not only acts at viral replication forks as an EBV DNA Pol processivity factor but also is widely distributed on newly synthesized EBV genomic DNA. Thus, the structures where the BMRF1 proteins were stained represent the locations of the newly synthesized viral genomes.
Viral replication proteins cluster to the replication compartments after induction of the lytic program. To gain further insight into the nuclear organization of lytic viral replication proteins, we examined their localization in lytic program-induced cells by immunostaining with viral replication protein-specific antibodies. The immunostaining was performed after extraction with 0.5%TX100-mCSK buffer to determine the localization of the active fractions (Fig. 4). The anti-BMRF1 and -BRLF1 protein specific antibodies are mouse monoclonal antibodies, and the other antibodies are rabbit polyclonal antibodies.
All of the cells that were positive for the BMRF1 protein were also positive for the BZLF1 protein, but not vice versa, especially early postinduction. The BZLF1 oriLyt binding protein showed a diffuse fine-granular pattern of distribution throughout the nuclei when the BMRF1 protein was not expressed in the immediate-early stages of induction (Fig. 4A, panels a). After 24 h postinduction, the BZLF1 foci, the intensity of which increased with time, appeared clearly in the replication compartments where BMRF1 proteins were stained uniformly (Fig. 4A, panels b). The fine-granular staining of the BZLF1 proteins was detected precisely within the same intranuclear globular structures where the BMRF1 proteins were localized, demonstrating that the mature globular structures contained both BZLF1 and BMRF1 proteins. The only difference is that the BZLF1 protein showed a fine-granular staining pattern, whereas the BMRF1 staining was homogenous.
The BALF2 single-stranded-DNA binding proteins were also codistributed as distinct spots within the BMRF1 protein-localized replication compartments (Fig. 4A, panels c and d). The BALF5 Pol catalytic protein and BBLF2/3 helicase-primase-associated protein also codistributed as distinct spots within the BMRF1 protein-localized replication compartments (Fig. 4A, panels f and g). Since these viral replication proteins are thought to act at viral replication forks, the foci may represent sites of viral replication factories, reminiscent of cellular replication factories containing PCNA, a homotrimeric clamp at replication forks that has been well studied (17).
To demonstrate that the BZLF1 behaves in a similar manner in a more natural reactivation setting, lytic infection was induced in B95-8 cells by treating the cells with TPA and sodium butyrate. As shown in Fig. 4B, panel b, the BZLF1 protein was distributed within the replication compartments with a diffuse fine-granular pattern of staining. Also, the BALF2 proteins were localized within the replication compartments as distinct spots (Fig. 4B, panels a). Thus, these observations are consistent with those for Tet-BZLF1/B95-8 cells and eliminate a concern that overexpression of the BZLF1 protein distorts the subcellular localization of viral replication proteins.
The BZLF1 and BRLF1 proteins are not recruited to the replication compartments at an early replication stage. The BRLF1 immediate-early transcription factor, which is also essential for viral DNA replication (5), was also clustered to the replication compartments (Fig. 4A, panels e). At an early replication stage when the replication compartments were not formed, the BRLF1 protein showed a diffuse fine-granular pattern of distribution throughout the nuclei, as did the BZLF1 protein (Fig. 5A and B, panels a). As the lytic infection progressed, the BRLF1 foci, the intensity of which increased with time, appeared clearly in the replication compartments where the BMRF1 proteins were stained uniformly (Fig. 5B). The staining patterns of the BZLF1 and BRLF1 proteins within the replication compartments were almost same. Bell et al. reported that the BRLF1 protein usually does not completely colocalize with the EBV genomes at early replication stages but becomes colocalized at the later stages (1), in agreement with our observation.
Inhibition of viral replication by phosphonoacetic acid, a viral DNA polymerase inhibitor, eliminates the DNA-bound form of the BMRF1 protein. The herpesvirus DNA polymerase inhibitor PAA does not inhibit chromosomal DNA replication at all but prevents viral DNA replication (15). In lytic infection, when viral DNA replication is blocked by the addition of PAA, viral prereplicative sites represented by staining of the BALF2 protein are formed (1). Lytic program-induced Tet-BZLF1/B95-8 cells were treated with or without 0.5%TX100-mCSK buffer and then doubly stained with anti-BALF2 and anti-BMRF1 specific antibodies in the presence or absence of PAA (Fig. 6A). As described above, in the absence of PAA, both the BMRF1 and BALF2 proteins were resistant to detergent treatment and colocalized at replication compartments (Fig. 6A, panels a and b). In the presence of PAA, the BALF2 protein was synthesized and localized in nuclei as scattered spots (Fig. 6A, panels c and d), although neither BrdU incorporation nor EBV DNA marked by FISH was detected (Fig. 3A and B, panels d). The scattered spots of BALF2 protein are reminiscent of prereplication sites formed during herpes simplex virus type 1 infection in the presence of PAA (27). The BALF2 protein might bind not to newly synthesized viral DNA but to originally existent viral genomic DNAs under these conditions. On the other hand, the BMRF1 protein was removed after treatment with 0.5%TX100-mCSK buffer, and little localized at the BALF2 protein-localized sites in the presence of PAA. The BMRF1 protein was sufficiently expressed and distributed diffusely throughout nuclei when the cells were stained without detergent treatment (compare Fig. 6A, panels c and d). As shown in Fig. 6B, biochemical fractionation analyses revealed that almost all of the BMRF1 protein expressed in the presence of PAA was in the non-DNA-bound form, while half of the BALF2 protein was in the detergent-resistant form, consistent with the results of the confocal microscopy analyses. These observations strongly support the idea that the BMRF1 protein binds to newly replicated EBV DNA at replication compartments.
In the presence of PAA, the immediate-early BZLF1 and BRLF1 proteins were distributed throughout nuclei and were not recruited to the BALF2 protein-localized sites (Fig. 6C). Thus, when the viral DNA synthesis is blocked by PAA, no clustering of the viral replication proteins to discrete sites within nuclei is observed.
Active fractions of the BALF5, BALF2, and BBLF2/3 proteins are associated at replication factories in the replication compartments. The BALF5 DNA polymerase, the BALF2 single-stranded-DNA binding protein, and the EBV helicase-primase complex conceivably work together at replication forks to synthesize leading and lagging strands of the concatemeric EBV genome during lytic infection (22). The BBLF2/3 protein is a component of the viral helicase-primase complex (8, 29). To further investigate spatial relationship among them, coimmunostaining analyses were performed. Since the anti-BALF5, -BALF2, and -BBLF2/3 protein specific antibodies are all rabbit polyclonal antibodies, the lytic program-induced Tet-BZLF1/B95-8 cells treated with 0.5%TX100-mCSK buffer were first reacted with anti-BALF2 or -BBLF2/3 primary antibody, followed by Alexa Fluor 488-conjugated secondary antibody, and then were finally stained with the anti-BALF5 protein antibody directly conjugated with Alexa 594.
As shown in Fig. 7, the distinct dots of the BALF2 single-stranded-DNA binding protein completely coincided with those of the BALF5 Pol protein within the replication compartments. Also, the BBLF2/3 proteins were codistributed with the BALF5 protein. In contrast, the staining pattern of the BZLF1 protein within the replication compartments was totally different from that of the BALF5 proteins (Fig. 7). Thus, it was clearly demonstrated that the EBV DNA polymerase, single-stranded-DNA binding protein, and helicase-primase complex cooperate at viral replication factories in the replication compartments, probably representing replication forks on the replicating EBV genome.
DISCUSSION
Establishment of the Tet-BZLF1/B95-8 cell system allowed us to precisely analyze the architecture of the replication compartments formed during EBV lytic infection. We could directly demonstrate that after induction of lytic replication, EBV replication proteins are clustered to locations of newly replicated EBV DNA in nuclei (namely, replication compartments) by the combination of FISH, BrdU incorporation, and immunostaining of viral replication proteins.
Our data indicate that the BZLF1 proteins are associated with replicating EBV genomes in the replication compartments throughout lytic infection. This may be via BZLF1 binding sites in oriLyt and immediate-early and early promoter regions of EBV genomes. It was reported that oriLyt is attached to the nuclear matrix after induction of the lytic cycle (19). We also observed that oriLyt sequences remained in the nonchromatin nuclear structures and nuclear matrix (data not shown). Our data obtained by biochemical fractionation revealed that the levels of DNase I-resistant BZLF1 protein were increased with the progression of lytic replication, although most of the BZLF1 proteins bind to EBV DNA solubilized by DNase I treatment (Fig. 2). It is possible that some BZLF1 proteins remaining in the insoluble nonchromatin nuclear structure fraction could be an actual active fraction binding to oriLyt. Further study will be needed to determine this.
Once the viral lytic replication origin is unwound by an unknown mechanism, viral replication proteins initiate to synthesize the viral genome. Our biochemical and immunostaining data suggest that active fractions of the BALF5 Pol catalytic proteins and BALF2 single-stranded-DNA binding proteins are actually associated with replication forks on the replicating EBV genome. These active fractions of viral replication proteins were resistant to detergent treatment, suggesting that these are in DNA-bound forms. The distinct dots of the BALF5 proteins distributed within replication compartments may represent viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures (3, 13, 14), viral replication factories are easily solubilized by DNase I treatment. Cellular DNA replication should be strictly controlled in relation to other nuclear events such as transcription. Under such conditions, highly organized domain structures based on nuclear matrix would be required. Compared with that situation, viral replication would be simpler so that construction of replication domains would be more relaxed.
In contrast to the case for the BZLF1, BALF5, BBLF2/3, and BALF2 proteins, the BMRF1 protein showed a homogenous distribution in the replication compartment. The protein is expressed abundantly in lytic replication-induced cells, and the expressed proteins are in a DNA-bound form. The BMRF1 protein is a viral Pol processivity factor (24), and thus it may also act at replication forks together with the BALF5 polymerase and BALF2 single-stranded-DNA binding proteins. Nevertheless, they showed a homogenous, not dot-like, distribution. In the presence of PAA, the BALF2 single-stranded-DNA binding protein formed a number of distinct foci (prereplicative sites) throughout nuclei, while the BMRF1 protein was hardly detected on the sites. No newly replicated viral DNAs were observed on the prereplicative sites (Fig. 3). Collectively, these data suggest that the BMRF1 protein may be widely distributed on the newly synthesized EBV double-stranded DNA genome by its double-stranded-DNA binding activity. It might function to stabilize the synthesized viral genomic DNA during the lytic replication like histones protecting chromosomal DNA.
The replication foci detected by BrdU staining grew larger with the progression of lytic infection, finally forming kidney-shaped islands filling the nuclei. We speculate that the replication compartment grows by accretion while maintaining the same relative position within the nucleus and that the coalescence of the merging of neighboring replication compartments formed larger compartments that eventually filled the nucleus.
We observed that upon induction of the lytic program, viral replication proteins are clustered to discrete sites (replication compartments) where newly synthesized viral DNAs are accumulated. In other words, chromosomal DNA synthesis was totally blocked and lytic viral DNA replication occurred. However, some cells exhibited diffuse BrdU staining throughout nuclei (data not shown), representing cellular DNA synthesis. In such cells there was no expression of viral replication proteins at all. It might be possible that viral lytic replication does not occur in cells in which chromosomal DNA synthesis has already started in S phase.
Our data strongly suggest that the viral replication proteins tested interact with newly synthesized viral DNA genomes during lytic replication. In order to examine whether these virus-encoded proteins constitute part of virus particles, we have purified EBV virions from culture medium of lytic program-induced Tet-BZLF1/B95-8 cells treated with doxycycline for 5 days. The purified virus particles were subjected 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 any of these proteins in these fractions (data not shown), indicating that the viral replication proteins may dissociate from the viral genome by an unknown molecular mechanism when the DNAs are packaged into viral capsids in nuclei.
ACKNOWLEDGMENTS
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 (grants 14021138 and 12470073 to T.T. and grant 16590398 to T.D.).
T.D. and A.K. contributed equally to this work.
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Virology Division, National Cancer Center Research Institute, Chuohku, Tokyo, Japan
ABSTRACT
Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.
INTRODUCTION
Epstein-Barr virus (EBV) is a human herpesvirus that infects 90% of individuals. Primary EBV infection targets resting B lymphocytes, inducing continuous proliferation. In B-lymphoblastoid cell lines, only limited numbers of viral genes are usually expressed and there is no production of virus particles; this is called latent infection. In the latent state, EBV maintains its 170-kb genome as complete, multiple copies of plasmids. Latent-phase viral replication appears to faithfully mimic cellular replicons: EBV genomes or small oriP/EBNA-1 plasmids are synthesized only once in each S phase by the host cell replication machinery, following the rules of chromosome replication (28).
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 (7) and leads to an ordered cascade of viral gene expression: activation of early gene expression followed by the lytic cascade of viral genome replication and late gene expression.
Lytic replication differs from the latent amplification state in that multiple rounds of replication are initiated within oriLyt, and the replication process has a greater dependence on EBV-encoded replication proteins (12). In the viral productive cycle, the EBV genome is amplified more than 100-fold. Intermediates of viral DNA replication are found as large head-to-tail concatemeric molecules, probably resulting from rolling-circle DNA replication, which are subsequently cleaved into unit-length genomes and packaged into virions in the nucleus. The lytic phase of EBV DNA replication is dependent on seven viral replication proteins: the BZLF1 protein, an oriLyt binding protein; the BALF5 protein, a DNA polymerase (Pol); the BMRF1 protein, a Pol processivity factor; the BALF2 protein, a single-stranded-DNA binding protein; and the BBLF4, BSLF1, and BBLF2/3 proteins, which are predicted to be helicase, primase, and helicase-primase-associated proteins, respectively (6). It has been suggested that all except the BZLF1 protein conceivably work together at replication forks to synthesize leading and lagging strands of the concatemeric EBV genome (22).
It is generally accepted that nucleic acid metabolism, such as DNA replication and transcription, is carried out on spatiotemporally organized domain structures in the cell nucleus (16). Nonchromatin nuclear structures such as the nuclear matrix, the scaffold, and the nucleoskeleton have been suggested as key players in organizing high-order chromatin and nuclear structures (2, 3). In the case of DNA replication, for example, fluorescence microscopic analyses have revealed discrete granular sites of replication, i.e., replication sites or replication foci (17, 18). Replication foci may be constructed based on nonchromatin nuclear structures, since nascent DNA and many proteins involved in DNA synthesis have been found to attach to these (3, 13, 14). In the case of EBV lytic replication, it was previously demonstrated that the BZLF1 and BMRF1 proteins distribute diffusely in nuclei at the immediate-early stage and then redistribute and colocalize to common globular regions, called replication compartments, in the nuclei (20). Furthermore, it has recently been reported that upon lytic activation, interchromosomally located nuclear domain 10 becomes dispersed in the cells, and replicating EBV genomes were frequently found beside the nuclear domain (1). However, detailed analyses of the architecture of the replication compartments remain to be carried out.
We have previously established a biochemical fractionation method which enables us to detect the active fraction of cellular DNA replication initiation proteins that bind tightly to chromatin and nuclear matrix (10). Using this method, we have been studying the nuclear organization of the chromosomal initiation proteins and their spatiotemporal regulation (9, 10). In this study, taking advantage of this method and confocal microscopy analyses, we performed detailed and comprehensive analyses of the architecture of the replication compartments formed during EBV lytic replication.
MATERIALS AND METHODS
Cell culture. Tet-BZLF1/B95-8 cells (15) were maintained in RPMI medium supplemented with 1 μg of puromycin/ml, 250 μg of hygromycin B/ml, and 10% tetracycline-free fetal calf serum (FCS) (Clontech, Inc.). To induce lytic EBV replication, the tetracycline derivative doxycycline was added to the culture medium at a final concentration of 2 μg/ml. B95-8 cells were grown at 37°C in a 5% CO2 atmosphere in RPMI 1640 medium with 10% FCS. For induction of lytic replication in B95-8 cells, phorbol 12-myristate 13-acetate (TPA) at a final concentration of 100 ng/ml and 5 mM sodium n-butyrate were added to the culture medium. When blocking lytic DNA replication, phosphonoacetic acid (PAA), a herpesvirus DNA polymerase-specific inhibitor, was added to the culture medium at a final concentration of 400 μg/ml.
Antibodies. The anti-BRLF1 protein specific rabbit polyclonal antibody was prepared as follows. DNA fragments carrying the BRLF1 gene was amplified by PCR methods with a pair of primers, BRLF1-UP (5'-GGGAATTCGGGCATTTCCTCTGTTACTACTAGCCA-3') and BRLF1-DN (5'-TTAGCAATGCCTGTGGCTCATGCATAGTTT-3'). The DNA fragment containing a full-length copy of the BRLF1 gene was ligated between the EcoRI and HindIII sites downstream of the polyhedrin promoter region in pFastBac DUAL (Life Technologies). The method for the preparation of recombinant baculovirus was described previously (29). The BRLF1 protein was purified from Sf21 cells infected with the recombinant baculovirus AcBRLF1. The BRLF1 protein-specific rabbit polyclonal antibody was produced against the purified protein and was affinity purified with BRLF1 protein-coupled Sepharose 4B as described previously (10). The preparation and specificity of the anti-BZLF1 and -BALF2 rabbit polyclonal antibodies were described previously (26, 29). Anti-BALF5 specific rabbit antibody (25) was affinity purified with BALF5 protein-coupled Sepharose 4B as described previously (10). For double immunostaining with rabbit polyclonal antibodies, Alexa Fluor 594-conjugated anti-BALF5 protein specific rabbit antibody was prepared with Fluor dye materials, using the Alexa Fluor 594 protein labeling kit (Molecular Probes). After coupling, the Alexa Fluor 594-conjugated antibody was separated from free dye by gel filtration with Bio-Gel P-30 (Bio-Rad). Anti-BBLF2/3 specific rabbit antibody (29) was affinity purified with BBLF2/3 protein-coupled Sepharose 4B as described previously (10). An anti-BMRF1 specific monoclonal antibody, R3, and an anti-BRLF1 specific mouse monoclonal antibody were purchased from Chemicon and Argen Inc., respectively. The BZLF1 protein-specific mouse monoclonal antibody was purchased from Dako Inc. Alexa Fluor 488-conjugated antibromodeoxyuridine (anti-BrdU) mouse monoclonal antibody and secondary goat anti-rabbit or -mouse immunoglobulin G (IgG) antibodies conjugated with Alexa Fluor 488 or 594 were purchased from Molecular Probes.
Biochemical cellular fractionation and analysis of nucleus-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 were lysed for 10 min on ice with 1 ml of ice-cold 0.5%TX100-mCSK buffer [10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (pH 6.8), 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg of aprotinin per ml, 0.5% Triton X-100] containing multiple protease inhibitors (protease inhibitor mixture for mammalian cell extracts [Sigma], 25 μl/ml), 200 mM Na3VO4, and 20 mM NaF. The samples were then subjected to centrifugation (2,000 x g, 3 min, 4°C) to obtain Triton X-100-extractable fractions. The detergent-extracted nuclei were then digested with 250 U of DNase I (10 U/μl; Roche Molecular Biochemicals) per ml in 0.1%TX100-mCSK containing 2 mM MgCl2 at 25°C for 20 min. The samples were then centrifuged to obtain the solubilized chromatin fraction and the remaining nonchromatin nuclear structures. Each sample was adjusted to the same volume by adding 2x sodium dodecyl sulfate (SDS) sample buffer and boiled, and aliquots corresponding to 1.8 x 104 cells per lane was subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride membranes, and the proteins were detected with the enhanced chemiluminescence detection system (Amersham) as described previously (15). Images were processed by LumiVision PRO (Aisin/Taitec Inc.) with a cooled charge-coupled-device camera and assembled in an Apple G4 computer with Adobe Photoshop 5.0. Signal intensity was quantified with a LumiVision image analyzer.
Immunofluorescence analysis. All staining procedures except extraction were carried out at room temperature. For immunofluorescence experiments, newly synthesized DNAs were labeled by incubating Tet-BZLF1/B95-8 cells with 10 μM BrdU, 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 (Sigma; 25 μl/ml), 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% FCS in PBS and then incubated for 1 h with each primary antibody diluted in PBS containing 10% FCS or overnight with anti-BRLF1 rabbit polyclonal antibody at 4°C. The anti-BALF2, anti-BRLF1, anti-BZLF1, anti-BALF5, and anti-BBLF2/3 protein rabbit polyclonal antibodies were used at 5 μg/ml. The anti-BMRF1, anti-BZLF1, and anti-BRLF1 mouse monoclonal antibodies and the control IgG were also 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 Fluor 488 or 594. Neither of the control antibodies yielded specific signals in either single- or double-staining experiments.
For staining of BrdU-incorporated DNA, the cells were treated for 10 min with 2 N HCl containing 0.5% Triton X-100 to expose the incorporated BrdU residues before blocking. The cells were washed twice with PBS and neutralized with 0.1 M sodium tetraborate (pH 9.0) for 5 min. For the BrdU staining, Alexa Fluor 488-conjugated anti-BrdU 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 with a Bio-Rad Radiance 2000 confocal laser-scanning microscope equipped with a PlainApo 100x 1.4-numerical-aperture oil immersion objective lens. Images were processed and assembled in an Apple G4 computer with Adobe Photoshop 5.0.
Fluorescence in situ hybridization (FISH). The EBV BamHI-W fragment was labeled with Chroma Tide Alexa Fluor 594-5-dUTP (Molecular Probes, Inc.) by random primer labeling according to the procedure provided by the manufacturer and was used for the detection of amplified EBV genomes during lytic infection. The fragment was small enough to prevent the visualization of single episomes, thus enabling us to observe only EBV DNA that replicates upon induction of the lytic cycle, not latent EBV genomes.
Cells were fixed in 4% paraformaldehyde (PFA) in PBS (10 min at room temperature) or, when FISH was combined with immunocytochemistry, first immunostained as described above and then refixed in 4% PFA to cross-link bound antibodies. After being washed in PBS and permeabilized in 0.2% Triton X-100 (20 min on ice), cells were digested with RNase A (Qiagen) (100 μg/ml in PBS for 30 min at 37°C). The cells were then dehydrated in an ethanol series (70, 80, and 100% for 3 min each at 20°C), air dried, and immediately covered with a probe mixture containing 50% formamide in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), probe DNA (10 ng/μl), 10% dextran sulfate, salmon sperm DNA (0.1 μg/μl; Sigma), and yeast tRNA (1 μg/μl; Sigma). The probe and cells were simultaneously heated at 94°C for 4 min to denature DNA and incubated overnight at 37°C. After hybridization, specimens were washed at 37°C with 50% formamide in 2x SSC (twice for 15 min each) and 2x SSC (10 min). Finally, the cells were equilibrated in PBS and mounted in Vectashield (Vector Laboratories, Inc).
RESULTS
In a recent study we isolated latently EBV-infected Tet-BZLF1/B95-8 cells in which the exogenous BZLF1 protein is conditionally expressed under the control of a tetracycline-regulated promoter (15). As judged by flow cytometry analysis, more than 80% of Tet-BZLF1/B95-8 cells became positive for BZLF1 and BMRF1 proteins 24 h after doxycycline addition (15). The copy number of the viral genome in the cells was significantly amplified after 24 h postinduction (p.i.). After induction, the growth of the cells is completely arrested. Thus, the system is simple and highly efficient for conditional induction of the lytic replication program in the absence of any other external stimuli, allowing us to analyze precisely the architecture of the replication compartments of EBV lytic replication.
Biochemical fractionation to determine the subcellular localizations of EBV replication proteins during lytic infection. We have previously determined the subcellular localizations of mammalian chromosomal DNA replication initiation proteins such as origin recognition complex (Orc), CDC6, and minichromosome maintenance protein complex (MCM) in HeLa cells by using biochemical fractionation methods (9, 10). These proteins assemble sequentially to form prereplication chromatin. In this method, HeLa cells are first treated with a buffer containing the nonionic detergent Triton X-100 in relatively physiological conditions, which extract not only cytoplasmic proteins but also nuclear proteins that are not tightly bound to nuclear structures. The proteins remaining in the extracted nuclei have indeed been shown to bind to chromatin and nuclear matrix and thus represent functionally active fractions. Based on the findings obtained by this and other methods, we have suggested the spatiotemporal regulation of these initiation proteins (9, 10). For example, it has been demonstrated that human MCM2 to -7 proteins bind to chromatin as heterohexameric forms in G1 phase and dissociate as replication proceeds (11). In this study, we applied this method for analyzing the behavior of EBV replication proteins.
We first examined whether the method could be applied to analyses of Tet-BZLF1/B95-8 B-lymphoblastoid cells. Accordingly, Tet-BZLF1/B95-8 cells were treated with 0.5%TX100-mCSK buffer, and then the extracted nuclei were digested with DNase I to remove the bulk of chromatin and EBV genome. As control markers for subcellular localization, we employed the CDC6, MCM4, and MCM7 proteins, which are known to be components of the prereplication complex of chromosomal DNA replication. As shown in Fig. 1, about two-thirds of the MCM4 and MCM7 proteins were extracted with the buffer, and most of the remainder was solubilized after DNase I treatment, demonstrating their chromatin binding. On the other hand, almost all of the CDC6 protein was detected in the non-detergent-extractable fraction, but in contrast to the chromatin-bound MCM proteins, the nucleus-bound CDC6 was resistant to DNase I treatment (Fig. 1). Thus, the CDC6 protein was in a nuclear matrix-bound form. These data are consistent with those previously obtained with HeLa cells (9, 10), confirming the reproducibility of the fractionation procedure with Tet-BZLF1/B95-8 cells. It should be noted that sufficient amounts of multiple protease inhibitors should be added to the extraction buffer in order to inhibit proteolysis as much as possible.
To investigate the subcellular distribution of EBV-encoded replication proteins after induction of the lytic program, lytic replication was induced in Tet-BZLF1/B95-8 cells with doxycycline. Cells were harvested at the indicated times, and biochemical fractionation was performed. The kinetics of expression profiles of EBV replication proteins have been previously examined by immunoblot analysis (15). Before induction, expression of the lytic viral replication proteins was almost undetectable (Fig. 2). The BZLF1 protein became detectable at 4 h p.i. (data not shown) and reached a plateau at 48 h p.i. The other immediate-early protein, the BRLF1 protein, also appeared at 6 h p.i. with a plateau at 48 h p.i. Both the BZLF1 and BRLF1 proteins were strongly expressed at 24 h p.i. and continuously expressed to 72 h p.i. Biochemical fractionation analyses, as shown in Fig. 2, revealed that all the BZLF1 protein was detected in the non-detergent-extracted fraction and that the levels of the DNA-bound form, solubilized by DNase I treatment, increased with the progression of lytic replication. Like the BZLF1 protein, most of the BRLF1 proteins tightly bound to nuclei, and half of them were released by DNase I throughout lytic infection. These observations suggest that the BZLF1 and BRLF1 immediate-early proteins may be associated with the EBV genome throughout productive replication. Although from only these data we could not exclude the possibility that they also bind to cellular DNA, further immunostaining data indicated that this is not the case (see below).
The BALF2 single-stranded-DNA binding protein, the BALF5 polymerase catalytic protein, and the BMRF1 Pol accessory protein appeared after 12 h p.i. (data not shown) and reached a plateau at 24 h p.i. In contrast to the BZLF1 and BRLF1 proteins, about half of the BALF2 single-stranded-DNA binding protein and BALF5 Pol catalytic protein was extracted with 0.5%TX100-mCSK buffer at 24 h p.i. These fractions could be the excess proteins that do not function in situ. Most of the remainder was solubilized after DNase I treatment at 24 h p.i. (Fig. 2). The levels of the DNA-bound forms of both proteins increased as lytic replication proceeded (Fig. 2, 48 and 72 h). These fractions may be associated with replication forks on the replicated EBV genome. This notion was further supported by immunostaining data as described below.
As was reported previously (23), extremely large amounts of the BMRF1 proteins were found to be expressed in the lytic program-induced cells compared with the BALF5 Pol catalytic proteins as judged by Coomassie blue staining (data not shown). Almost all of the BMRF1 protein existed as a non-detergent-extractable DNA-bound form (Fig. 2), suggesting that the BMRF1 proteins might be widely associated with the EBV genome besides acting at viral replication forks as polymerase processivity factors. Again, this notion was supported by immunostaining data (see below).
The BMRF1 protein is localized in intranuclear focal sites of newly synthesized viral DNAs during lytic replication. Switching from the latent phase to viral productive replication causes complete arrest of cell cycle progression (15). Therefore, labeling with the thymidine analog BrdU and subsequent immunofluorescence analyses with confocal microscopy can be used to identify sites of viral DNA replication in B95-8 cells during lytic infections. Accordingly, the lytic program-induced Tet-BZLF1/B95-8 cells were labeled with BrdU for 1 h prior to harvesting and extracted with 0.5%TX100-mCSK buffer. It should be noted that this treatment strips non-DNA-bound form of viral or cellular proteins, making it useful to investigate the localization of functional fractions of viral replication proteins, as shown in Fig. 3. The cells were then fixed with methanol and stained with anti-BrdU rabbit antibody and anti-BMRF1 mouse monoclonal antibody after treatment with HCl for 10 min. The acid treatment little affected the distribution of BMRF1 and other proteins. We tested for specificity of the second antibodies and for reliability of discrimination with fluorescence microscopy filters. When cells were stained singly for either antigen with inappropriate combinations of first and second antibodies, no fluorescence was observed. Also, no immunofluorescence was observed with alternate filter.
BrdU staining appeared as small foci in the nuclei at early stages of viral DNA replication (Fig. 3A, upper panels). The staining pattern of BrdU, representing newly replicated DNA, completely coincided with that of the BMRF1 Pol accessory protein (Fig. 3A). Such globular structures were identified as the sites of EBV DNA localized in nuclei by FISH with a viral DNA probe (Fig. 3B), confirming the previous observations reported by Takagi et al. (20). Under the conditions used for FISH, we could not detect any signal of latently replicated viral genomes (data not shown). These sites of viral DNA synthesis have been termed replication compartments. The numbers of the spots representing replication compartments at early stages of the lytic replication were more than 10, which is lower than the average copy number (40 to 50 copies per cell) of the latent virus genome (Fig. 3A, upper panels). It is likely that only some of the latent viral genomes may become templates for lytic replication. With progression of lytic replication, the replication compartments became larger and appeared to fuse to form large globular structures that eventually filled the nucleus at late stages (Fig. 3), consistent with observations with herpes simplex virus type 1 (4, 21). As described above, the staining pattern of the BMRF1 Pol accessory protein completely coincided with the BrdU staining and localization of EBV DNA in the replication compartments (Fig. 3). Therefore, although the BMRF1 protein possesses an intrinsic double-stranded-DNA binding activity (23), it may bind to viral DNA preferentially and not to cellular chromosomal DNA. Furthermore, in the replication compartments, the BMRF1 proteins were distributed diffusely and homogenously, rather than as fine dots as was the case for the other viral replication proteins (see below). Thus, the immunostaining data together with the findings that almost all of the abundantly expressed BMRF1 protein molecules bound to DNA (Fig. 2A) indicate that BMRF1 not only acts at viral replication forks as an EBV DNA Pol processivity factor but also is widely distributed on newly synthesized EBV genomic DNA. Thus, the structures where the BMRF1 proteins were stained represent the locations of the newly synthesized viral genomes.
Viral replication proteins cluster to the replication compartments after induction of the lytic program. To gain further insight into the nuclear organization of lytic viral replication proteins, we examined their localization in lytic program-induced cells by immunostaining with viral replication protein-specific antibodies. The immunostaining was performed after extraction with 0.5%TX100-mCSK buffer to determine the localization of the active fractions (Fig. 4). The anti-BMRF1 and -BRLF1 protein specific antibodies are mouse monoclonal antibodies, and the other antibodies are rabbit polyclonal antibodies.
All of the cells that were positive for the BMRF1 protein were also positive for the BZLF1 protein, but not vice versa, especially early postinduction. The BZLF1 oriLyt binding protein showed a diffuse fine-granular pattern of distribution throughout the nuclei when the BMRF1 protein was not expressed in the immediate-early stages of induction (Fig. 4A, panels a). After 24 h postinduction, the BZLF1 foci, the intensity of which increased with time, appeared clearly in the replication compartments where BMRF1 proteins were stained uniformly (Fig. 4A, panels b). The fine-granular staining of the BZLF1 proteins was detected precisely within the same intranuclear globular structures where the BMRF1 proteins were localized, demonstrating that the mature globular structures contained both BZLF1 and BMRF1 proteins. The only difference is that the BZLF1 protein showed a fine-granular staining pattern, whereas the BMRF1 staining was homogenous.
The BALF2 single-stranded-DNA binding proteins were also codistributed as distinct spots within the BMRF1 protein-localized replication compartments (Fig. 4A, panels c and d). The BALF5 Pol catalytic protein and BBLF2/3 helicase-primase-associated protein also codistributed as distinct spots within the BMRF1 protein-localized replication compartments (Fig. 4A, panels f and g). Since these viral replication proteins are thought to act at viral replication forks, the foci may represent sites of viral replication factories, reminiscent of cellular replication factories containing PCNA, a homotrimeric clamp at replication forks that has been well studied (17).
To demonstrate that the BZLF1 behaves in a similar manner in a more natural reactivation setting, lytic infection was induced in B95-8 cells by treating the cells with TPA and sodium butyrate. As shown in Fig. 4B, panel b, the BZLF1 protein was distributed within the replication compartments with a diffuse fine-granular pattern of staining. Also, the BALF2 proteins were localized within the replication compartments as distinct spots (Fig. 4B, panels a). Thus, these observations are consistent with those for Tet-BZLF1/B95-8 cells and eliminate a concern that overexpression of the BZLF1 protein distorts the subcellular localization of viral replication proteins.
The BZLF1 and BRLF1 proteins are not recruited to the replication compartments at an early replication stage. The BRLF1 immediate-early transcription factor, which is also essential for viral DNA replication (5), was also clustered to the replication compartments (Fig. 4A, panels e). At an early replication stage when the replication compartments were not formed, the BRLF1 protein showed a diffuse fine-granular pattern of distribution throughout the nuclei, as did the BZLF1 protein (Fig. 5A and B, panels a). As the lytic infection progressed, the BRLF1 foci, the intensity of which increased with time, appeared clearly in the replication compartments where the BMRF1 proteins were stained uniformly (Fig. 5B). The staining patterns of the BZLF1 and BRLF1 proteins within the replication compartments were almost same. Bell et al. reported that the BRLF1 protein usually does not completely colocalize with the EBV genomes at early replication stages but becomes colocalized at the later stages (1), in agreement with our observation.
Inhibition of viral replication by phosphonoacetic acid, a viral DNA polymerase inhibitor, eliminates the DNA-bound form of the BMRF1 protein. The herpesvirus DNA polymerase inhibitor PAA does not inhibit chromosomal DNA replication at all but prevents viral DNA replication (15). In lytic infection, when viral DNA replication is blocked by the addition of PAA, viral prereplicative sites represented by staining of the BALF2 protein are formed (1). Lytic program-induced Tet-BZLF1/B95-8 cells were treated with or without 0.5%TX100-mCSK buffer and then doubly stained with anti-BALF2 and anti-BMRF1 specific antibodies in the presence or absence of PAA (Fig. 6A). As described above, in the absence of PAA, both the BMRF1 and BALF2 proteins were resistant to detergent treatment and colocalized at replication compartments (Fig. 6A, panels a and b). In the presence of PAA, the BALF2 protein was synthesized and localized in nuclei as scattered spots (Fig. 6A, panels c and d), although neither BrdU incorporation nor EBV DNA marked by FISH was detected (Fig. 3A and B, panels d). The scattered spots of BALF2 protein are reminiscent of prereplication sites formed during herpes simplex virus type 1 infection in the presence of PAA (27). The BALF2 protein might bind not to newly synthesized viral DNA but to originally existent viral genomic DNAs under these conditions. On the other hand, the BMRF1 protein was removed after treatment with 0.5%TX100-mCSK buffer, and little localized at the BALF2 protein-localized sites in the presence of PAA. The BMRF1 protein was sufficiently expressed and distributed diffusely throughout nuclei when the cells were stained without detergent treatment (compare Fig. 6A, panels c and d). As shown in Fig. 6B, biochemical fractionation analyses revealed that almost all of the BMRF1 protein expressed in the presence of PAA was in the non-DNA-bound form, while half of the BALF2 protein was in the detergent-resistant form, consistent with the results of the confocal microscopy analyses. These observations strongly support the idea that the BMRF1 protein binds to newly replicated EBV DNA at replication compartments.
In the presence of PAA, the immediate-early BZLF1 and BRLF1 proteins were distributed throughout nuclei and were not recruited to the BALF2 protein-localized sites (Fig. 6C). Thus, when the viral DNA synthesis is blocked by PAA, no clustering of the viral replication proteins to discrete sites within nuclei is observed.
Active fractions of the BALF5, BALF2, and BBLF2/3 proteins are associated at replication factories in the replication compartments. The BALF5 DNA polymerase, the BALF2 single-stranded-DNA binding protein, and the EBV helicase-primase complex conceivably work together at replication forks to synthesize leading and lagging strands of the concatemeric EBV genome during lytic infection (22). The BBLF2/3 protein is a component of the viral helicase-primase complex (8, 29). To further investigate spatial relationship among them, coimmunostaining analyses were performed. Since the anti-BALF5, -BALF2, and -BBLF2/3 protein specific antibodies are all rabbit polyclonal antibodies, the lytic program-induced Tet-BZLF1/B95-8 cells treated with 0.5%TX100-mCSK buffer were first reacted with anti-BALF2 or -BBLF2/3 primary antibody, followed by Alexa Fluor 488-conjugated secondary antibody, and then were finally stained with the anti-BALF5 protein antibody directly conjugated with Alexa 594.
As shown in Fig. 7, the distinct dots of the BALF2 single-stranded-DNA binding protein completely coincided with those of the BALF5 Pol protein within the replication compartments. Also, the BBLF2/3 proteins were codistributed with the BALF5 protein. In contrast, the staining pattern of the BZLF1 protein within the replication compartments was totally different from that of the BALF5 proteins (Fig. 7). Thus, it was clearly demonstrated that the EBV DNA polymerase, single-stranded-DNA binding protein, and helicase-primase complex cooperate at viral replication factories in the replication compartments, probably representing replication forks on the replicating EBV genome.
DISCUSSION
Establishment of the Tet-BZLF1/B95-8 cell system allowed us to precisely analyze the architecture of the replication compartments formed during EBV lytic infection. We could directly demonstrate that after induction of lytic replication, EBV replication proteins are clustered to locations of newly replicated EBV DNA in nuclei (namely, replication compartments) by the combination of FISH, BrdU incorporation, and immunostaining of viral replication proteins.
Our data indicate that the BZLF1 proteins are associated with replicating EBV genomes in the replication compartments throughout lytic infection. This may be via BZLF1 binding sites in oriLyt and immediate-early and early promoter regions of EBV genomes. It was reported that oriLyt is attached to the nuclear matrix after induction of the lytic cycle (19). We also observed that oriLyt sequences remained in the nonchromatin nuclear structures and nuclear matrix (data not shown). Our data obtained by biochemical fractionation revealed that the levels of DNase I-resistant BZLF1 protein were increased with the progression of lytic replication, although most of the BZLF1 proteins bind to EBV DNA solubilized by DNase I treatment (Fig. 2). It is possible that some BZLF1 proteins remaining in the insoluble nonchromatin nuclear structure fraction could be an actual active fraction binding to oriLyt. Further study will be needed to determine this.
Once the viral lytic replication origin is unwound by an unknown mechanism, viral replication proteins initiate to synthesize the viral genome. Our biochemical and immunostaining data suggest that active fractions of the BALF5 Pol catalytic proteins and BALF2 single-stranded-DNA binding proteins are actually associated with replication forks on the replicating EBV genome. These active fractions of viral replication proteins were resistant to detergent treatment, suggesting that these are in DNA-bound forms. The distinct dots of the BALF5 proteins distributed within replication compartments may represent viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures (3, 13, 14), viral replication factories are easily solubilized by DNase I treatment. Cellular DNA replication should be strictly controlled in relation to other nuclear events such as transcription. Under such conditions, highly organized domain structures based on nuclear matrix would be required. Compared with that situation, viral replication would be simpler so that construction of replication domains would be more relaxed.
In contrast to the case for the BZLF1, BALF5, BBLF2/3, and BALF2 proteins, the BMRF1 protein showed a homogenous distribution in the replication compartment. The protein is expressed abundantly in lytic replication-induced cells, and the expressed proteins are in a DNA-bound form. The BMRF1 protein is a viral Pol processivity factor (24), and thus it may also act at replication forks together with the BALF5 polymerase and BALF2 single-stranded-DNA binding proteins. Nevertheless, they showed a homogenous, not dot-like, distribution. In the presence of PAA, the BALF2 single-stranded-DNA binding protein formed a number of distinct foci (prereplicative sites) throughout nuclei, while the BMRF1 protein was hardly detected on the sites. No newly replicated viral DNAs were observed on the prereplicative sites (Fig. 3). Collectively, these data suggest that the BMRF1 protein may be widely distributed on the newly synthesized EBV double-stranded DNA genome by its double-stranded-DNA binding activity. It might function to stabilize the synthesized viral genomic DNA during the lytic replication like histones protecting chromosomal DNA.
The replication foci detected by BrdU staining grew larger with the progression of lytic infection, finally forming kidney-shaped islands filling the nuclei. We speculate that the replication compartment grows by accretion while maintaining the same relative position within the nucleus and that the coalescence of the merging of neighboring replication compartments formed larger compartments that eventually filled the nucleus.
We observed that upon induction of the lytic program, viral replication proteins are clustered to discrete sites (replication compartments) where newly synthesized viral DNAs are accumulated. In other words, chromosomal DNA synthesis was totally blocked and lytic viral DNA replication occurred. However, some cells exhibited diffuse BrdU staining throughout nuclei (data not shown), representing cellular DNA synthesis. In such cells there was no expression of viral replication proteins at all. It might be possible that viral lytic replication does not occur in cells in which chromosomal DNA synthesis has already started in S phase.
Our data strongly suggest that the viral replication proteins tested interact with newly synthesized viral DNA genomes during lytic replication. In order to examine whether these virus-encoded proteins constitute part of virus particles, we have purified EBV virions from culture medium of lytic program-induced Tet-BZLF1/B95-8 cells treated with doxycycline for 5 days. The purified virus particles were subjected 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 any of these proteins in these fractions (data not shown), indicating that the viral replication proteins may dissociate from the viral genome by an unknown molecular mechanism when the DNAs are packaged into viral capsids in nuclei.
ACKNOWLEDGMENTS
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 (grants 14021138 and 12470073 to T.T. and grant 16590398 to T.D.).
T.D. and A.K. contributed equally to this work.
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