| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Graduate Institute and Department of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan 100, PR China
Correspondence
Mei-Ru Chen
mrc{at}ntu.edu.tw
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
These authors contributed equally to this work. 
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). To maintain genome integrity, mammalian cells have evolved various processes to monitor and repair damaged DNA. DNA damage may trigger cell-cycle-checkpoint activation, transcriptional activation, repair mechanisms, damage tolerance or even apoptosis, if the damage is severe (Friedberg, 2001). The repair mechanisms include recombination repair, mismatch repair (MMR), base-excision repair (BER) and nucleotide-excision repair (NER). Double-stranded breaks in DNA are usually recognized by the MRN (Mre11–Rad50–NBS1) complex and the activated ataxia telangiectasia mutated/related (ATM/ATR) pathway. The binding of the MRN complex activates cellular damage machinery through ATM and ATR (Shiloh, 2003). The post-replicative MMR system plays important roles in removing base–base and small insertion–deletion mismatches that arise during DNA replication (Kunkel & Erie, 2005). BER is the major process for removing a damaged or inappropriate base. Hydrolysis of the N-glycosidic bond creates an abasic site in the DNA, allowing AP endonuclease 1 to generate a nick, for polymerase to fill in the correct nucleotide through short-patch BER or via long-patch BER, which depends on replication proteins (Krokan et al., 2002). NER is used widely to remove many types of DNA lesions, such as DNA damage caused by exposure to UV light and bulky DNA induced by chemicals (Friedberg, 2001).
The basic steps of NER include damage recognition, dual incision and release of a short damaged region (24–32 nt), repair synthesis to fill the gap, and ligation (Friedberg, 2001). Two subpathways are distinguishable in the repair synthesis and ligation process: transcription-coupled NER (TC-NER), which removes damaged DNA located on the transcribed strand, and global genomic NER (GG-NER), which repairs lesions throughout genomic DNA. For GG-NER, xeroderma pigmentosum C (XPC) binds tightly to hHR23B to perform the damage recognition. The binding of the XPC–hHR23B complex triggers the binding of XPA, TFIIH, XPG, RPA and ERCC1–XPF. The ERCC1–XPF complex then serves as a duplex/single-stranded DNA endonuclease to cut the damaged strand at the 5' junction, whilst XPG cuts the damaged strand at the 3' junction to release the short damaged fragment to initiate the subsequent repair process (Friedberg, 2001).
Under such stringent control of cellular DNA fidelity, viral DNA replication within the nucleus strongly elicits host DNA-repair systems. Viruses thus need to inactivate the DNA signalling pathway or modify host repair or replication factors to facilitate replication of their genomes (Lu & Chen, 2006). The cellular DNA damage-sensor MRN complex, ATM and BER proteins have been shown to be recruited to viral replication compartments (Weitzman et al., 2004).
Epstein–Barr virus (EBV) is a widely distributed 
-herpesvirus that infects about 90 % of the population worldwide. Following primary infection, it becomes latent in the B lymphocytes of most individuals. Reactivation of EBV can be induced by chemical agents, UV light or surface IgG cross-linking (Rickinson & Kieff, 2001). Viral DNA replication initiates at oriLyt, with the viral DNA replication core machinery containing seven virus-encoded proteins: BZLF1 (an oriLyt-binding protein), BALF5 (DNA polymerase), BMRF1 (also called EA-D, a polymerase processivity factor), BALF2 (a single-stranded-DNA-binding protein), BBLF4 (helicase), BSLF1 (primase) and BBLF2/3 (helicase–primase-associated protein) (Fixman et al., 1995). During lytic replication, the cellular DNA-repair response may be stimulated because of the accumulation of abundant viral DNA. Phosphorylated ATM and MRN complex are recruited to the EBV replication compartment (Kudoh et al., 2005). Although the cellular DNA-damage response is activated by lytic virus replication, p53 signalling downstream of the ATM checkpoint signal is blocked to prevent cell-cycle arrest and apoptosis. Moreover, the levels of cyclin E/A and the hyperphosphorylated form of RB are increased during lytic infection. This S-phase-like environment may benefit EBV lytic replication (Kudoh et al., 2005). Recently, it was demonstrated that proliferation cell nuclear antigen (PCNA), the clamp loader RF-C and MMR proteins MSH-2, MSH-6, MLH1 and hPSM2 are recruited to EBV replication compartments, suggesting that host MMR might function to increase viral genome integrity (Daikoku et al., 2006). Our previous study on viral and cellular uracil–DNA glycosylase also indicated that the cellular BER machinery may supplement viral DNA replication in cell culture (Lu et al., 2007). The interplay between viral and cellular DNA replication and repair machineries may play a pivotal role in EBV replication.
BGLF4 kinase is the only Ser/Thr protein kinase of EBV and phosphorylates several viral substrates, including DNA polymerase processivity factor BMRF1, EBNA-2, EBNA-LP and BZLF1 (Asai et al., 2006; Chen et al., 2000; Kato et al., 2003; Yue et al., 2005). It also phosphorylates cellular translation elongation factor 1 delta (EF-1
) and casein kinase II
at cellular cdc2-targeted sites (Kawaguchi et al., 2003). BGLF4 localizes within the viral replication compartment and is packaged into virions (Wang et al., 2005), indicating that BGLF4 may be involved in viral lytic DNA replication and virion egress. Indeed, viral lytic DNA replication was decreased by 30 % and viral nucleocapsids were retained in the nuclei when the expression of BGLF4 was knocked down by small interfering RNA (siRNA) (Gershburg et al., 2007). Interestingly, BGLF4 also phosphorylates MCM4 at sites inactivating DNA helicase activity of the MCM4–6–7 complex, which might lead to the blocking of chromosomal DNA replication in virus-reactivated cells (Kudoh et al., 2006). Recently, we have also observed that expression of BGLF4 can induce premature chromosome condensation through a cdc2-independent mechanism (Lee et al., 2007).
In this study, BGLF4 protein was shown, through yeast two-hybrid screening, to interact with XPC. This interaction was confirmed by co-immunoprecipitation and immunofluorescence assay in HeLa and EBV-positive NA cells. Functionally, BGLF4 stimulates cellular repair activity in host-cell reactivation in an XPC-dependent manner. By using an siRNA approach, XPC was shown to be required for efficient viral genome replication, suggesting that the cellular NER machinery also may contribute to viral genome integrity during its replication.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The bait plasmid pGBDU-BGLF4(S300P), containing a spontaneous mutation at aa 300, was used to screen a cDNA library of the human lymphocyte MATCHMAKER (Clontech) fused to the GAL4-AD in pACT. The bait plasmid and the cDNA library were transformed sequentially into the yeast strain PJ69-4A (MATa trp1-901 leu2-3, 112 ura3-52 his3-200 gal4
gal80 LYS2 : : GAL1-HIS3 GAL2-ADE2 met2 : : GAL7-lacZ) (James et al., 1996). The transformants were then plated on synthetic complete medium lacking adenine (Ade), uracil (Ura) and leucine (Leu). The Ade+/Ura+/Leu+ colonies were purified and the cDNA clones were isolated, sequenced and subjected to a BLAST search of GenBank.
Plasmids.
Wild-type BGLF4 (pYPW17) and catalytic-site mutant (‘kinase-dead’) BGLF4 (K102I, pYPW20) are pSG5-based constructs (Wang et al., 2005). To express glutathione S-transferase (GST) fusion protein, BamHI–EcoRI fragments containing BGLF4 ORFs from pYWP17 or pYPW20 were modified to be in frame with GST and cloned into pGEX3X (Pharmacia Biotech). The p53-expressing plasmid (pCMV-p53) and pCMV-Luc, which contains a Renilla luciferase gene driven by the cytomegalovirus (CMV) promoter, have been described previously by Liu et al. (2004). The XPC-expressing plasmid (pcDNA3.1B/XPCwt; a gift from Dr Wenya Huang, National Cheng-Kung University, Taiwan) has been described previously by Chang et al. (2003). The Rta-expressing plasmid RTS15 (pSG5-Rta, a gift from Dr Diane Hayward, Johns Hopkins School of Medicine, Baltimore, MD, USA) was described by Ragoczy & Miller (1999). The siGFP plasmid is the pSUPER-based construct described by Chang et al. (2004). The siRNA-expressing plasmids (siXPC-1, siXPC-2 and siXPC-3) were constructed by cloning siRNA sequences, which were predicted using an online program (http://www.sirnawizard.com/siRNA.php), into pSUPER via the BamHI and HindIII sites. The siRNA sequences were subjected to a BLAST search against the human genome and EST databases to ensure that no other human genes were targeted. The siXPC-1, siXPC-2 and siXPC-3 RNAs are directed against the XPC sequences 5'-GAGCCTTCTCTCCAAAGTT-3' (nt 208–226 of the cDNA), 5'-GAAGACAAGAGAAAGAAGT-3' (nt 590–608 of the cDNA) and 5'-GAGGAATTGGTCCATATAT-3' (nt 957–975 of the cDNA), respectively.
Cell culture and induction of the viral lytic cycle.
H1299/bcl2 cells (Liu et al., 2004) had been established previously by transfecting a plasmid expressing bcl-2 into H1299 cells, a human large cell lung carcinoma cell line without the p53 gene (Mitsudomi et al., 1992). NPC-TW01 is an NPC cell line lacking the EBV genome (Lin et al., 1993) and the NA cell line was derived from NPC-TW01 and converted with a neomycin-containing recombinant Akata EBV (Chang et al., 1999). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum. XP4PA-SV cells, which carry a deletion mutation in the xpc gene (Chang et al., 2003), were obtained from Dr Wenya Huang and maintained in DMEM supplemented with 15 % fetal calf serum. For induction of the EBV lytic cycle, NA cells were transfected with the Rta-expressing plasmid RTS15.
Antibodies.
The primary antibodies used for the immunoprecipitation assay, immunoblotting and indirect immunofluorescence included Rta mAb 467, Zta mAb 4F10 (Tsai et al., 1997), EA-D mAb 88A9 (Tsai et al., 1991), BGLF4 mAb 2224 (Wang et al., 2005), XPC mAb (Abcam), -actin mAb (Sigma), HA mAb (Covance) and rabbit antisera against BGLF4 (Wang et al., 2005).
Immunoprecipitation assay and immunoblotting.
HA-XPC- and BGLF4-expressing plasmids were transfected into 293T cells by using the calcium phosphate method (Chen & Okayama, 1987). At 48 h post-transfection, cells were lysed in NP-40 lysis buffer [50 mM Tris/HCl (pH 8.0), 120 mM NaCl and 0.5 % Nonidet P-40]. To detect the interaction of BGLF4 and XPC in virus-replicating cells, Rta-, BGLF4- and XPC-expressing plasmids were cotransfected into NA cells by using Lipofectamine 2000 (Invitrogen). At 36 h post-transfection, cells were lysed in NP-40 lysis buffer containing 100 mM sodium fluoride, 2 mM sodium vanadate and 1xComplete Mini Protease Inhibitor mixture (EDTA-free; Roche). Lysates (1.5 mg total protein) were precleared with protein A–Sepharose CL-4B beads (Amersham Biosciences) at 4 °C for 1 h. The supernatants were incubated with anti-BGLF4 mAb 2224 (Wang et al., 2005) or anti-HA mAb at 4 °C for 1.5 h. The immunocomplexes were precipitated with protein A–Sepharose beads at 4 °C for 1.5 h and washed extensively with PBS. Finally, the immunoprecipitates were separated by SDS-PAGE and transferred onto Hybond-C super membrane (Amersham Biosciences). The blot was incubated with the primary antibodies indicated and then with horseradish peroxidase-labelled secondary antibodies. After incubation, the blot was developed with substrate (ECL Western blotting; Amersham Biosciences) and the luminescence was detected by exposure to X-ray film.
Expression and purification of GST–BGLF4 fusion protein.
Rosetta-gami (DE3) competent cells (Novagen) were transformed with GST–BGLF4- or GST–K102I-expressing plasmids and colonies were inoculated into Luria–Bertani (LB) medium containing 100 µg ampicillin ml–1, 50 µg chloramphenicol ml–1, 50 µg kanamycin ml–1 and 50 µg tetracycline ml–1, and incubated overnight in a shaking incubator. The cultures were diluted 1 : 50 into LB medium containing 100 µg ampicillin ml–1, 50 µg chloramphenicol ml–1, 50 µg kanamycin ml–1, 50 µg tetracycline ml–1 and 10 % (w/v) glucose, and grown with vigorous shaking at 37 °C for 4 h. The cultures were kept on ice for 30 min. IPTG was then added to a final concentration of 0.5 mM and the cultures were incubated overnight at 24 °C in a shaking incubator. The bacteria were harvested by centrifugation, resuspended in ice-cold PBS containing 1 % Triton X-100, 1xComplete Mini Protease Inhibitor Mixture (EDTA-free; Roche) and 1 µg lysozyme ml–1, incubated on ice for 1 h and lysed by sonication at 4 °C. The extracts were purified by using a glutathione column.
GST pull-down assay.
In vitro transcription/translation was performed by using a TNT rabbit reticulocyte lysate (Promega) with pcDNA3.1B/XPCwt in the presence of [35S]methionine. A 30 µl volume of [35S]methionine-labelled in vitro-transcribed and -translated XPC protein was incubated with a 50 % slurry of glutathione–Sepharose beads and 3 µg GST or GST–BGLF4 fusion protein in lysis buffer [20 mM Tris–Cl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 0.5 % Nonidet P-40 and 1xComplete Mini Protease Inhibitor mixture] for 2 h at 4 °C with rotation. The mixtures were centrifuged at maximum speed (16 100 g) for 2 min at 4 °C in a microfuge. The beads were washed four times with 1 ml ice-cold lysis buffer. Beads were boiled, resolved by SDS-PAGE and analysed by autoradiography.
Indirect immunofluorescence.
To detect colocalization of XPC and BGLF4, HeLa or NA cells were slide-cultured, transfected with the XPC- and BGLF4- or Rta-expressing plasmids, harvested at the time points indicated, air-dried, fixed with 4 % paraformaldehyde in PBS for 30 min and permeabilized for 5 min with 0.1 % Triton X-100. To solubilize DNA-unbound forms of viral or cellular proteins, the protocol was adapted from that described by Kudoh et al. (2005). Briefly, cells were treated with 0.5 % Triton X-100–mCSK buffer [10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM PMSF, 10 µg aprotinin ml–1 and 0.5 % Triton X-100] for 10 min on ice, followed by fixation with 70 % methanol for 20 min on ice and washing with PBS. Immunostaining was conducted with mouse anti-XPC antibody (1 : 200; Abcam) and rabbit anti-BGLF4 antibody 2224 (1 : 100) at 37 °C for 1.5 h. Rhodamine red-conjugated anti-mouse IgG antibodies (1 : 100; Jackson) and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody (1 : 100; Jackson) were used as secondary antibodies. After washing with PBS, cells were stained with Hoechest 33258 at room temperature for 1 min and covered with mounting medium (H1000; Vector) for fluorescence microscopy (Axioskop 40 FL; Zeiss).
Detection and quantification of EBV DNA.
For quantification of EBV DNA, real-time PCR was performed according to the manufacturer's instructions (Applied Biosystems). Briefly, cells were lysed, digested with proteinase K and then subjected to real-time PCR analysis as described previously (Chang et al., 2002). The EBNA1 region of the EBV genome was the detection target for real-time PCR and the primers and probes have been described previously (Lo et al., 1999). Endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA, which served as a control, was quantified by using specific primers (Chang et al., 2002) and Universal Probe Library probe 045 (Roche). The H2B4 cell line, harbouring one EBV genome per cell, was used to generate a standard curve for quantification and the EBV copy number was calculated by comparison with the standards (Chang et al., 2002). All samples were tested in duplicate and the experiments were repeated twice independently.
Host-cell reactivation (HCR) assay.
The HCR assay protocol was adapted from that described by Liu et al. (2004). Briefly, a firefly luciferase reporter plasmid (pCMV-Luc), either damaged with 1000 or 100 J UV light m–2 (Stratalinker UV Cross-linker 1800) as indicated or mock-treated, together with an undamaged Renilla luciferase reporter plasmid (pWP-1) as an internal control, were cotransfected with effector plasmids BGLF4, BGLF4 (K102I), p53 or XPC into H1299/bcl2 or XP4PA-SV cells. At 24 or 48 h post-transfection, the cells were harvested and subjected to a luciferase assay with a Dual-Glo assay kit (Promega) according to the manufacturer's instructions. The activity of Renilla luciferase (from the internal control) was used to calibrate the activity of firefly luciferase (from the reporter). Repair activity was determined from two parameters, repair conversion and fold HCR. Repair conversion was calculated first by dividing normalized luciferase activity from cells transfected with UV light-irradiated pCMV-Luc by that of non-irradiated pCMV-Luc transfectants. The repair conversion of vector transfectants was set to 1. The fold HCR was then calculated by comparing the repair conversion of effector to that of vector transfectants.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). As illustrated in Fig. 1(a), aa 509–835 of XPC overlap partially with the interacting domain for hHR23B and TFIIH. The interaction of XPC (aa 509–835) with full-length (S300P) BGLF4, its N terminus (aa 1–293) and C terminus (aa 201–429) was reproduced in a yeast two-hybrid system (data not shown), suggesting that XPC may interact with either a domain within the aa 201–293 region or separate domains in the N and C termini of BGLF4. To confirm the interaction of BGLF4 with XPC in mammalian cells, a ‘kinase-dead’ version of BGLF4 (K102I) and HA–XPC (aa 509–835) were cotransfected into 293T cells for co-immunoprecipitation assays with either anti-BGLF4 or anti-HA antibody. HA–XPC (aa 509–835) was precipitated specifically from BGLF4 (K102I) and HA–XPC (aa 509–835)-cotransfected 293T cells by using anti-BGLF4 antibody (Fig. 1b, lane 1). This interaction also was detected in a reciprocal co-immunoprecipitation assay performed with anti-HA antibody (Fig. 1b, lane 4). Moreover, full-length XPC also was co-immunoprecipitated from wild-type BGLF4 and XPC-cotransfected 293T cells using anti-BGLF4 antibody (Fig. 1c, lane 1). GST pull-down experiments further revealed that in vitro-expressed [35S]methionine-labelled XPC protein can bind to both GST–BGLF4 and GST–BGLF4 KD (Fig. 1d). Even though the signal is not very strong, these results indicate that BGLF4 interacts with XPC both in vitro and in vivo.
|
, colocalization of BGLF4 and XPC was observed in the nucleus by using an indirect immunofluorescence assay (IFA). BGLF4 has been shown to colocalize with the DNA polymerase processivity factor BMRF1 (EA-D) and the immediate-early transactivator Zta in the EBV lytic replication compartment during the early stages of replication (Asai et al., 2006; Wang et al., 2005). Therefore, we sought to determine whether XPC colocalizes with BGLF4 in the viral replication compartment. EBV-positive NA cells were cotransfected with plasmids expressing XPC and EBV immediate-early transactivator, Rta, which can induce EBV lytic DNA replication. Ectopic expression of Rta induced the expression of BGLF4 in NA cells, as detected by IFA (Fig. 2b). Moreover, XPC partially colocalized with BGLF4 in cells undergoing lytic DNA replication at 24, 36 and 48 h post-induction (p.i.). To confirm this observation further, the Rta- and XPC-cotransfected NA cells were treated with 0.5 % Triton X-100–mCSK buffer to solubilize DNA-unbound forms of BGLF4 and XPC proteins, and fixed (Fig. 2c). Similar colocalization patterns of BGLF4 and XPC were observed in the nuclei of detergent-treated cells, suggesting that at least a portion of XPC appears to localize to the viral replication compartment while viral genomes are synthesized.
|
), indicating that BGLF4 interacts specifically with XPC in NA cells undergoing lytic DNA replication.
|
). Amplification of EBV DNA was detected simultaneously by using quantitative real-time PCR (Fig. 4b). Expression of siXPC-1 and siXPC-3 resulted in around 30–40 % inhibition of Rta-induced amplification of viral genomes at 48 h p.i. In contrast, expression of siXPC-2, siGFP and pSUPER had no effect on Rta-induced amplification of the viral genome. This observation suggests that XPC may be required for increased efficiency of EBV DNA replication.
|
). Expression of ‘kinase-dead’ BGLF4 (K102I) did not enhance DNA-repair activity in H1299/bcl2 cells (Fig. 5b), suggesting that BGLF4 enhances DNA-repair activity in a kinase activity-dependent and p53-independent manner.
|
). As expected, ectopic expression of XPC in XP4PA-SV cells enhanced DNA repair in a dose-dependent manner (Fig. 5d; data not shown). In the presence of 0.015 µg XPC plasmid, the XPC-mediated DNA-repair activity was enhanced further by BGLF4 in a dose-dependent manner (Fig. 5d). Interestingly, expression of BGLF4 (K102I) also enhances DNA repair in an XPC-dependent manner in XP4PA-SV cells (Fig. 5c, d). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Although the interaction is not very strong, data presented here demonstrated repeatedly that BGLF4 could interact with XPC in vitro and in vivo. Because BGLF4 has also recently been demonstrated to interact with many viral and cellular factors in a yeast two-hybrid system (Calderwood et al., 2007), we speculate that BGLF4 may interact with different cellular and viral factors in cells and that this is why only a small portion of BGLF4 or protein was immunoprecipitated reciprocally. Secondly, XPC colocalizes and interacts with BGLF4 in virus-replicating NA cells (Figs 2 and 3). Thirdly, when siXPC1 and siXPC3 were used to knock down XPC, EBV lytic DNA replication was suppressed to 60–70 % of the level observed for the vector control (Fig. 4). To our knowledge, this is the first study to demonstrate that NER contributes to EBV lytic DNA replication.
Complex interactions among viral and cellular DNA-replication and -repair systems, contributing to efficient virus replication, have been reported in several studies of herpesviruses. By using immunoprecipitation and mass spectrometry, the single-stranded DNA-binding protein ICP8 of herpes simplex virus 1 (HSV-1) has previously been shown to be associated with over 50 cellular and viral proteins. Many of the co-immunoprecipitated cellular proteins are involved in DNA replication, recombination and repair, suggesting that HSV-1 recruits them to aid its replication or that they target abnormal viral DNA structures generated during replication (Taylor & Knipe, 2004). For example, the ATM signal-transduction pathway is activated in HSV-1-infected and EBV-reactivated cells (Kudoh et al., 2005; Shirata et al., 2005). In EBV-replicating cells, Mre11-Rad50-NBS1 (MRN) complexes recognize newly synthesized viral DNA and are recruited to sites of viral replication. Phosphorylated p53 is also recruited to the replication compartment and interacts with Zta. However, p53 downstream signalling is blocked; therefore, the virus can actively promote an S-phase-like environment to facilitate its replication (Kudoh et al., 2005). Lytic infection of HSV-1 induces NBS-1 phosphorylation and recruits NBS-1 to viral precursors of replication compartments, suggesting that the activation component of ATM signalling is also beneficial for efficient virus infection (Shirata et al., 2005). Overall, various mistakes generated during viral DNA replication may rely on cellular machineries to provide repair activities.
In addition, PCNA and components of mismatch repair (MMR) factors, including MSH-2, MSH-6, MLH-1 and hPSM2, are recruited to the EBV lytic replication compartment. Their recruitment might be required for PCNA to load onto newly synthesized viral DNA (Daikoku et al., 2006). Recently, our study also indicated that cellular UNG2 and EBV BKRF3-encoded viral uracil-DNA glycosylase (UDG) activities play an important role in viral lytic DNA replication (Lu et al., 2007). UDG is the primary DNA repair enzyme of the BER pathway responsible for removal of inappropriate uracil from DNA. Moreover, cellular UDG (UNG2) can interact and colocalize with PCNA and replication protein A (RPA) in cellular replication foci (Otterlei et al., 1999). BKRF3 also is associated with BGLF4 and viral DNA polymerase (BALF5) in the viral replication compartment (Lu et al., 2007). In addition, depletion of cellular UNG2 in Kaposi's sarcoma-associated herpesvirus-positive cells using short hairpin RNA (shRNA) reduced the number of copies of the viral genome (Verma et al., 2006). Regarding the role of the DNA-repair system in viral DNA replication, NER is shown here for the first time, to our knowledge, to contribute to EBV lytic DNA replication. Notably, RPA associated with XPC to recognize DNA lesions in NER. PCNA has also been shown previously to be required for NER for the DNA-resynthesis step in vitro (Shivji et al., 1992). This study reveals that XPC interacts with and colocalizes with BGLF4 in EBV-positive cells undergoing viral lytic DNA replication (Figs 1, 2 and 3). Considering these observations together, we postulate that the interaction of BGLF4 and XPC can recruit cellular replication and repair proteins such PCNA and RPA to the viral replication compartment to enhance replication efficiency. This suggestion is partly supported by the recent observation that knockdown of BGLF4 using siRNA reduced viral DNA synthesis to about 70 % (Gershburg et al., 2007).
Several virally encoded proteins have been shown to regulate NER activity. The hepatitis B virus oncoprotein HBx suppresses both global and transcription-coupled NER (Groisman et al., 1999; Mathonnet et al., 2004). Human T-cell leukemia virus type 1 (HTLV-1) transactivator and transforming protein Tax has also been shown to inhibit BER and NER (Kao & Marriott, 1999; Philpott & Buehring, 1999). In this study, we observed that expression of BGLF4 stimulated the DNA-repair activity of exogenous UV-damaged substrates in an HCR assay (Fig. 5a). The enhancement was not observed in XPC-defective XP4PA-SV cells, indicating that BGLF4 enhances DNA repair through an XPC-dependent pathway in vivo (Fig. 5b, c). Notably, BGLF4 could enhance DNA-repair activity in both a p53-dependent and -independent manner, and kinase activity was not required to stimulate HCR in p53-positive cells. We suspect that BGLF4 may function as a recruitment protein and may also provide some DNA damage signalling simultaneously. In NER, p53 can modulate the helicase activity of XPB and XPD, regulate the expression of XPC and serve as a chromatin-accessibility factor for NER of DNA damage (Adimoolam & Ford, 2002; Rubbi & Milner, 2003; Wang et al., 1995), whereas BGLF4 can interact with XPC and topoisomerase II (Lee et al., 2007). Such interactions might be expected to somehow enhance DNA-repair efficiency through recruitment of proteins for formation of more stable complexes. Within p53-negative cells, because BGLF4 is known to phosphorylate cdk1 and cdk2 substrates, it is possible that BGLF4 may phosphorylate some unknown cellular factors participating in the DNA-repair signalling pathway, thereby enhancing the DNA-repair activity. This may also explain why, in the presence of p53, the kinase activity of BGLF4 was not required to enhance DNA-repair activity. Overall, we suggest that BGLF4 may recruit XPC to the viral replication compartment, enhance NER activity and thereby promote efficient production of viral DNA.
In summary, we demonstrated that the viral kinase BGLF4 interacts specifically and colocalizes with the critical NER factor XPC in vivo. Furthermore, BGLF4 enhances XPC-mediated DNA-repair activity through p53-dependent and -independent mechanisms. Inhibition of XPC expression results in suppression of EBV lytic DNA replication, indicating that XPC might play an important role in EBV lytic DNA replication. Our results suggest that, in addition to BER, MMR and double-stranded DNA repair, NER also contributes to EBV DNA replication, although the specific mechanisms have yet to be identified. Future insights into the mechanisms of interplay of cellular DNA repair and viral DNA replication should allow a more complete understanding of EBV lytic cycle replication control and the pathogenesis of EBV-associated malignancies.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Asai, R., Kato, A., Kato, K., Kanamori-Koyama, M., Sugimoto, K., Sairenji, T., Nishiyama, Y. & Kawaguchi, Y. (2006). Epstein–Barr virus protein kinase BGLF4 is a virion tegument protein that dissociates from virions in a phosphorylation-dependent process and phosphorylates the viral immediate-early protein BZLF1. J Virol 80, 5125–5134.
Calderwood, M. A., Venkatesan, K., Xing, L., Chase, M. R., Vazquez, A., Holthaus, A. M., Ewence, A. E., Li, N., Hirozane-Kishikawa, T. & other authors (2007). Epstein–Barr virus and virus human protein interaction maps. Proc Natl Acad Sci U S A 104, 7606–7611.
Chang, Y., Tung, C. H., Huang, Y. T., Lu, J., Chen, J. Y. & Tsai, C. H. (1999). Requirement for cell-to-cell contact in Epstein–Barr virus infection of nasopharyngeal carcinoma cells and keratinocytes. J Virol 73, 8857–8866.
Chang, Y., Cheng, S. D. & Tsai, C. H. (2002). Chromosomal integration of Epstein–Barr virus genomes in nasopharyngeal carcinoma cells. Head Neck 24, 143–150.[CrossRef][Medline]
Chang, H. C., Tsai, J., Guo, Y. L., Huang, Y. H., Tsai, H. N., Tsai, P. C. & Huang, W. (2003). Differential UVC-induced gadd45 gene expression in xeroderma pigmentosum cells. Biochem Biophys Res Commun 305, 1109–1115.[CrossRef][Medline]
Chang, Y., Chang, S. S., Lee, H. H., Doong, S. L., Takada, K. & Tsai, C. H. (2004). Inhibition of the Epstein–Barr virus lytic cycle by Zta-targeted RNA interference. J Gen Virol 85, 1371–1379.
Chen, C. & Okayama, H. (1987). High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7, 2745–2752.
Chen, M. R., Chang, S. J., Huang, H. & Chen, J. Y. (2000). A protein kinase activity associated with Epstein–Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro. J Virol 74, 3093–3104.
Daikoku, T., Kudoh, A., Sugaya, Y., Iwahori, S., Shirata, N., Isomura, H. & Tsurumi, T. (2006). Postreplicative mismatch repair factors are recruited to Epstein–Barr virus replication compartments. J Biol Chem 281, 11422–11430.
Fixman, E. D., Hayward, G. S. & Hayward, S. D. (1995). Replication of Epstein–Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays. J Virol 69, 2998–3006.[Abstract]
Friedberg, E. C. (2001). How nucleotide excision repair protects against cancer. Nat Rev Cancer 1, 22–33.[CrossRef][Medline]
Gershburg, E., Raffa, S., Torrisi, M. R. & Pagano, J. S. (2007). Epstein–Barr virus-encoded protein kinase (BGLF4) is involved in production of infectious virus. J Virol 81, 5407–5412.
Groisman, I. J., Koshy, R., Henkler, F., Groopman, J. D. & Alaoui-Jamali, M. A. (1999). Downregulation of DNA excision repair by the hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cells. Carcinogenesis 20, 479–483.
Hoeijmakers, J. H. (2001). Genome maintenance mechanisms for preventing cancer. Nature 411, 366–374.[CrossRef][Medline]
James, P., Halladay, J. & Craig, E. A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–1436.[Abstract]
Kao, S. Y. & Marriott, S. J. (1999). Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein. J Virol 73, 4299–4304.
Kato, K., Yokoyama, A., Tohya, Y., Akashi, H., Nishiyama, Y. & Kawaguchi, Y. (2003). Identification of protein kinases responsible for phosphorylation of Epstein–Barr virus nuclear antigen leader protein at serine-35, which regulates its coactivator function. J Gen Virol 84, 3381–3392.
Kawaguchi, Y., Kato, K., Tanaka, M., Kanamori, M., Nishiyama, Y. & Yamanashi, Y. (2003). Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1. J Virol 77, 2359–2368.
Krokan, H. E., Drablos, F. & Slupphaug, G. (2002). Uracil in DNA – occurrence, consequences and repair. Oncogene 21, 8935–8948.[CrossRef][Medline]
Kudoh, A., Fujita, M., Zhang, L., Shirata, N., Daikoku, T., Sugaya, Y., Isomura, H., Nishiyama, Y. & Tsurumi, T. (2005). Epstein–Barr virus lytic replication elicits ATM checkpoint signal transduction while providing an S-phase-like cellular environment. J Biol Chem 280, 8156–8163.
Kudoh, A., Daikoku, T., Ishimi, Y., Kawaguchi, Y., Shirata, N., Iwahori, S., Isomura, H. & Tsurumi, T. (2006). Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4–MCM6–MCM7 complex during Epstein–Barr virus productive replication. J Virol 80, 10064–10072.
Kunkel, T. A. & Erie, D. A. (2005). DNA mismatch repair. Annu Rev Biochem 74, 681–710.[CrossRef][Medline]
Lee, C. P., Chen, J. Y., Wang, J. T., Kimura, K., Takemoto, A., Lu, C. C. & Chen, M. R. (2007). Epstein–Barr virus BGLF4 kinase induces premature chromosome condensation through activation of condensin and topoisomerase II. J Virol 81, 5166–5180.
Lin, C. T., Chan, W. Y., Chen, W., Huang, H. M., Wu, H. C., Hsu, M. M., Chuang, S. M. & Wang, C. C. (1993). Characterization of seven newly established nasopharyngeal carcinoma cell lines. Lab Invest 68, 716–727.[Medline]
Liu, M. T., Chen, Y. R., Chen, S. C., Hu, C. Y., Lin, C. S., Chang, Y. T., Wang, W. B. & Chen, J. Y. (2004). Epstein–Barr virus latent membrane protein 1 induces micronucleus formation, represses DNA repair and enhances sensitivity to DNA-damaging agents in human epithelial cells. Oncogene 23, 2531–2539.[CrossRef][Medline]
Lo, Y. M., Chan, L. Y., Chan, A. T., Leung, S. F., Lo, K. W., Zhang, J., Lee, J. C., Hjelm, N. M., Johnson, P. J. & Huang, D. P. (1999). Quantitative and temporal correlation between circulating cell-free Epstein–Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 59, 5452–5455.
Lu, C.-C. & Chen, M.-R. (2006). Lytic replication of Epstein–Barr virus. Future Virol 1, 435–446.[CrossRef]
Lu, C. C., Huang, H. T., Wang, J. T., Slupphaug, G., Li, T. K., Wu, M. C., Chen, Y. C., Lee, C. P. & Chen, M. R. (2007). Characterization of the uracil-DNA glycosylase activity of Epstein–Barr virus BKRF3 and its role in lytic viral DNA replication. J Virol 81, 1195–1208.
Mathonnet, G., Lachance, S., Alaoui-Jamali, M. & Drobetsky, E. A. (2004). Expression of hepatitis B virus X oncoprotein inhibits transcription-coupled nucleotide excision repair in human cells. Mutat Res 554, 305–318.[Medline]
Mitsudomi, T., Steinberg, S. M., Nau, M. M., Carbone, D., D'Amico, D., Bodner, S., Oie, H. K., Linnoila, R. I., Mulshine, J. L. & other authors (1992). p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features. Oncogene 7, 171–180.[Medline]
Otterlei, M., Warbrick, E., Nagelhus, T. A., Haug, T., Slupphaug, G., Akbari, M., Aas, P. A., Steinsbekk, K., Bakke, O. & Krokan, H. E. (1999). Post-replicative base excision repair in replication foci. EMBO J 18, 3834–3844.[CrossRef][Medline]
Philpott, S. M. & Buehring, G. C. (1999). Defective DNA repair in cells with human T-cell leukemia/bovine leukemia viruses: role of tax gene. J Natl Cancer Inst 91, 933–942.
Ragoczy, T. & Miller, G. (1999). Role of the Epstein–Barr virus RTA protein in activation of distinct classes of viral lytic cycle genes. J Virol 73, 9858–9866.
Rickinson, A. B. & Kieff, E. (2001). Epstein–Barr virus and its replication. In Fields Virology, 4th edn, pp. 2575–2627. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Rubbi, C. P. & Milner, J. (2003). p53 is a chromatin accessibility factor for nucleotide excision repair of DNA damage. EMBO J 22, 975–986.[CrossRef][Medline]
Shiloh, Y. (2003). ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 3, 155–168.[CrossRef][Medline]
Shirata, N., Kudoh, A., Daikoku, T., Tatsumi, Y., Fujita, M., Kiyono, T., Sugaya, Y., Isomura, H., Ishizaki, K. & Tsurumi, T. (2005). Activation of ataxia telangiectasia-mutated DNA damage checkpoint signal transduction elicited by herpes simplex virus infection. J Biol Chem 280, 30336–30341.
Shivji, K. K., Kenny, M. K. & Wood, R. D. (1992). Proliferating cell nuclear antigen is required for DNA excision repair. Cell 69, 367–374.[CrossRef][Medline]
Taylor, T. J. & Knipe, D. M. (2004). Proteomics of herpes simplex virus replication compartments: association of cellular DNA replication, repair, recombination, and chromatin remodeling proteins with ICP8. J Virol 78, 5856–5866.
Tsai, C. H., Williams, M. V. & Glaser, R. (1991). Characterization of two monoclonal antibodies to Epstein–Barr virus diffuse early antigen which react to two different epitopes and have different biological function. J Virol Methods 33, 47–52.[CrossRef][Medline]
Tsai, C. H., Liu, M. T., Chen, M. R., Lu, J., Yang, H. L., Chen, J. Y. & Yang, C. S. (1997). Characterization of monoclonal antibodies to the Zta and DNase proteins of Epstein–Barr virus. J Biomed Sci 4, 69–77.[CrossRef][Medline]
Uchida, A., Sugasawa, K., Masutani, C., Dohmae, N., Araki, M., Yokoi, M., Ohkuma, Y. & Hanaoka, F. (2002). The carboxy-terminal domain of the XPC protein plays a crucial role in nucleotide excision repair through interactions with transcription factor IIH. DNA Repair (Amst) 1, 449–461.[CrossRef][Medline]
Verma, S. C., Bajaj, B. G., Cai, Q., Si, H., Seelhammer, T. & Robertson, E. S. (2006). Latency associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus. J Virol 80, 11178–11190.
Wang, X. W., Yeh, H., Schaeffer, L., Roy, R., Moncollin, V., Egly, J. M., Wang, Z., Freidberg, E. C., Evans, M. K. & other authors (1995). p53 modulation of TFIIH-associated nucleotide excision repair activity. Nat Genet 10, 188–195.[Medline]
Wang, J. T., Yang, P. W., Lee, C. P., Han, C. H., Tsai, C. H. & Chen, M. R. (2005). Detection of Epstein–Barr virus BGLF4 protein kinase in virus replication compartments and virus particles. J Gen Virol 86, 3215–3225.
Weitzman, M. D., Carson, C. T., Schwartz, R. A. & Lilley, C. E. (2004). Interactions of viruses with the cellular DNA repair machinery. DNA Repair (Amst) 3, 1165–1173.[CrossRef][Medline]
Yue, W., Gershburg, E. & Pagano, J. S. (2005). Hyperphosphorylation of EBNA2 by Epstein–Barr virus protein kinase suppresses transactivation of the LMP1 promoter. J Virol 79, 5880–5885.
Received 4 June 2007;
accepted 25 July 2007.
This article has been cited by other articles:
|
|
 |
|
  J.-T. Wang, S.-L. Doong, S.-C. Teng, C.-P. Lee, C.-H. Tsai, and M.-R. Chen Epstein-Barr Virus BGLF4 Kinase Suppresses the Interferon Regulatory Factor 3 Signaling Pathway J. Virol., February 15, 2009; 83(4): 1856 - 1869. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-P. Lee, Y.-H. Huang, S.-F. Lin, Y. Chang, Y.-H. Chang, K. Takada, and M.-R. Chen Epstein-Barr Virus BGLF4 Kinase Induces Disassembly of the Nuclear Lamina To Facilitate Virion Production J. Virol., December 1, 2008; 82(23): 11913 - 11926. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
