Role of ATM in the Formation of the Replication ... - Journal of Virology

Role of ATM in the Formation of the Replication Compartment during Lytic Replication of Epstein-Barr Virus in Nasopharyngeal Epithelial Cells Pok Man Hau,a* Wen Deng,a,b Lin Jia,a Jie Yang,a Tatsuya Tsurumi,c Alan Kwok Shing Chiang,d Michael Shing-Yan Huen,e Sai Wah Tsaoa Department of Anatomy and Center for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SARa; School of Nursing, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SARb; Division of Virology, Aichi Cancer Center Research Institute, Nagoya, Japanc; Department of Pediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SARd; Genome Stability Research Laboratory, Department of Anatomy and Centre for Cancer Research, The University of Hong Kong, Hong Kong SARe

ABSTRACT

Epstein-Barr virus (EBV), a type of oncogenic herpesvirus, is associated with human malignancies. Previous studies have shown that lytic reactivation of EBV in latently infected cells induces an ATM-dependent DNA damage response (DDR). The involvement of ATM activation has been implicated in inducing viral lytic gene transcription to promote lytic reactivation. Its contribution to the formation of a replication compartment during lytic reactivation of EBV remains poorly defined. In this study, the role of ATM in viral DNA replication was investigated in EBV-infected nasopharyngeal epithelial cells. We observed that induction of lytic infection of EBV triggers ATM activation and localization of DDR proteins at the viral replication compartments. Suppression of ATM activity using a small interfering RNA (siRNA) approach or a specific chemical inhibitor profoundly suppressed replication of EBV DNA and production of infectious virions in EBV-infected cells induced to undergo lytic reactivation. We further showed that phosphorylation of Sp1 at the serine-101 residue is essential in promoting the accretion of EBV replication proteins at the replication compartment, which is crucial for replication of viral DNA. Knockdown of Sp1 expression by siRNA effectively suppressed the replication of viral DNA and localization of EBV replication proteins to the replication compartments. Our study supports an important role of ATM activation in lytic reactivation of EBV in epithelial cells, and phosphorylation of Sp1 is an essential process downstream of ATM activation involved in the formation of viral replication compartments. Our study revealed an essential role of the ATM-dependent DDR pathway in lytic reactivation of EBV, suggesting a potential antiviral replication strategy using specific DDR inhibitors. IMPORTANCE

Epstein-Barr virus (EBV) is closely associated with human malignancies, including undifferentiated nasopharyngeal carcinoma (NPC), which has a high prevalence in southern China. EBV can establish either latent or lytic infection depending on the cellular context of infected host cells. Recent studies have highlighted the importance of the DNA damage response (DDR), a surveillance mechanism that evolves to maintain genome integrity, in regulating lytic EBV replication. However, the underlying molecular events are largely undefined. ATM is consistently activated in EBV-infected epithelial cells when they are induced to undergo lytic reactivation. Suppression of ATM inhibits replication of viral DNA. Furthermore, we observed that phosphorylation of Sp1 at the serine-101 residue, a downstream event of ATM activation, plays an essential role in the formation of viral replication compartments for replication of virus DNA. Our study provides new insights into the mechanism through which EBV utilizes the host cell machinery to promote replication of viral DNA upon lytic reactivation.

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erpesviruses belong to a large family of DNA viruses that can switch between latent and lytic cycles in infected host cells. They are categorized into three subfamilies: alpha-, beta-, and gammaherpesviruses. While the default pathway of alpha- and betaherpesviruses is lytic infection, the gammaherpesviruses are more variable in their infection life cycles (1). In the gammaherpesvirus family, the two most studied gammaherpesviruses are Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). Upon infection, latent or lytic infection is established depending on the cellular context of the host cells infected by the viruses. EBV infects more than 90% of the world’s human population and is closely associated with human malignancies, including Burkitt’s lymphoma, Hodgkin’s lymphoma, undifferentiated nasopharyngeal carcinoma, and gastric carcinoma (2). While EBV readily establishes latent infection in B lymphocytes, infection of

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Received 18 May 2014 Accepted 10 October 2014 Accepted manuscript posted online 29 October 2014 Citation Hau PM, Deng W, Jia L, Yang J, Tsurumi T, Chiang AKS, Huen MS-Y, Tsao SW. 2015. Role of ATM in the formation of the replication compartment during lytic replication of Epstein-Barr virus in nasopharyngeal epithelial cells. J Virol 89:652– 668. doi:10.1128/JVI.01437-14. Editor: R. M. Longnecker Address correspondence to Sai Wah Tsao, [email protected]. * Present address: Pok Man Hau, Department of Anatomical and Cellular Pathology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.01437-14

Journal of Virology

January 2015 Volume 89 Number 1

ATM Activation during EBV Reactivation

primary oropharyngeal epithelial cells is primarily lytic in nature (3). It has been proposed that acute replication of EBV in infected oropharyngeal epithelium in vivo is the main source of virus in saliva for transmission (4). During latent infection of EBV, multiple copies of the EBV genome (around 170 kb in size) are maintained as circular, chromatin-like DNA structures called episomes. These EBV episomes replicate once in S phase in latently infected cells using the host DNA replication machinery (5, 6). During lytic infection, replication of the viral genome occurs in nuclear domains inside host cells termed replication compartments. Within these replication compartments, EBV genomes are amplified 100- to 1,000-fold (7). The intermediates of these replicating EBV DNA molecules form concatemers involving rollingcircle replication of viral DNA. The giant concatemeric DNA molecules are eventually cleaved into individual EBV genomes and packaged into infectious virions that are released for transmission (7, 8). Six core EBV replication proteins—EA-D (the processivity factor BMRF1), BALF2 (the single-stranded DNA binding protein), BALF5 (the viral polymerase), BBLF4 (the helicase), BSLF1 (the primase), and BBLF2/3 (the linker protein)—together with Zta (BZLF1), are crucial for lytic viral replication (9–12). They are localized at viral replication compartments during lytic replication in EBV-infected cells (10, 13). Zta, which is the product of the BZLF1 gene, functions as a transcription activator of other viral lytic genes and as an origin binding protein to the lytic origin of DNA replication (oriLyt) of the EBV episome during lytic replication (14). Zta binds to the oriLyt and acts as a platform to tether other viral replication proteins (7, 10, 15, 16). In addition, cellular factors are also recruited to the oriLyt, forming a scaffolding complex essential for the replication of viral DNA (17). In the past few years, different viruses have been reported to initiate the DNA damage response (DDR) during primary infection and lytic reactivation (18–22). The DNA damage response is a surveillance mechanism to monitor genome integrity and coordinate different aspects of cellular response, including cell cycle progression, gene transcription, and DNA repair. Among the DDR proteins, ATM is a major transducer of the DNA damage response in response to DNA double-strand breaks (23). It has been reported that ATM activation may have different roles in different phases of the life cycle of viral infection in cells (24–28). Using an inducible BZLF1 expression system, activation of the ATM-dependent DNA damage response was observed during lytic induction of the EBV-infected marmoset B cell line B95.8 (29). A recent report showed that the EBV viral kinase BGLF4 phosphorylates TIP60, a histone acetyltransferase, which then acetylates ATM, resulting in full activation of ATM for efficient viral replication during lytic reactivation (18). Another recent report showed that ATM activity is required for lytic induction of EBV-infected cells upon stimulation by a variety of DNA-damaging agents (30). A role for the DDR in viral replication has also been proposed in murine gammaherpesvirus 68 (␥HV68) infection in a primary mouse bone marrow macrophage model that induces H2AX phosphorylation by the virus-encoded kinase orf36 (an analog of the EBV-encoded kinase BGLF4) (31). Apparently, triggering of the DNA damage response during viral infection is an active process to foster viral replication. Interestingly, ATM activation was found to be independent of viral DNA replication and lytic cycle induction in a previous study (29). Therefore, the role of ATM activation in lytic reactivation of herpesvirus is still controversial.


Pilot studies of the structural components and their organization in the EBV replication compartment have been reported (13, 29, 32). Strikingly, the DDR factors, including activated ATM and the MRN complex (consisting of Mre11, Rad50, and Nbs1), have been found to localize at these viral replication compartments, suggesting a functional relationship between the viral DNA replication proteins and host DDR factors (29). While the importance of ATM signaling in regulating EBV lytic replication has been established by these studies, the functional role of ATM recruitment to the formation of the viral replication compartment remains unclear. In this study, we demonstrate that ATM activation is required for efficient viral DNA replication and production of infectious virions from EBV-infected nasopharyngeal epithelial cells. Furthermore, we demonstrate that the phosphorylation of the host transcription factor Sp1 plays an important role during lytic reactivation of EBV. Phosphorylation of Sp1 is ATM dependent, and it probably functions as an important scaffold protein to promote tethering of viral core replication proteins at replication compartments to facilitate lytic replication of EBV DNA. Our findings suggest a dynamic role of the host DNA damage response in lytic replication of EBV and highlight the ability of EBV to hijack the host cellular responses to facilitate amplification of its DNA inside infected epithelial cells. MATERIALS AND METHODS Cell culture, transfections, and reagents. HONE1-EBV cells and Daudi cells (a B lymphoblast cell line) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin (P/S). HONE1-EBV cells were established by infecting EBVnegative HONE1 cells with enhanced green fluorescent protein (EGFP)tagged EBV (strain Akata) (33–35). The cells were grown at 37°C in a 5% CO2 incubator. To induce EBV reactivation in HONE1-EBV cells, 12-Otetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (NaB) were added to the culture medium at final concentrations of 40 ng/ml and 3 mM, respectively. For plasmid and small interfering RNA (siRNA) transfection, cells were transfected with X-tremeGene HP DNA transfection reagent (Roche) and Oligofectamine (Invitrogen), respectively, according to the manufacturer’s instructions. The chemicals used in this study were purchased from Sigma-Aldrich unless otherwise stated. Cloning of the BZLF1 gene. PCR amplifications were performed with either PrimeStar HS with GC buffer (TaKaRa) or Platinum Pfx DNA polymerase (Invitrogen). Total RNA from EBV-infected B95.8 cells treated with TPA (40 ng/ml) to induce lytic reactivation was purified with TRIzol (Invitrogen). Five micrograms of total RNA was used to synthesize cDNA using the SuperScript II Reverse Transcriptase Kit (Invitrogen). For cloning of BZLF1, 2 ␮l of cDNA was used to perform PCR amplification. The PCR-amplified products were subcloned into the pcDNA3 vector. For FLAG-tagged BZLF1, forward primers with a FLAG tag sequence were used to perform PCR using the BZLF1-pcDNA3 plasmid as the template. The PCR products were subcloned into pcDNA3. Sanger DNA sequencing was used to verify the gene sequence (BGI-Tech, Hong Kong). RNA interference to silence ATM and Sp1 expression. All the siRNAs were ordered from GenePharma (Shanghai, China). Fifty nanomolar siRNA (the working concentration) was used in the experiments unless otherwise stated. The sequences of siRNA targeting ATM and Sp1 were as follows: siATM, sense, 5=-CAUACUACUCAAAGACAUUdTdT-3=, and antisense, 5=-AAUGUCUUUGAGUAGUAUGTT-3=; siSP1 (1), sense, 5=AUCAAAACUUAGAGCUACCdTdT-3=, and antisense, 5=-GGUAGCU CUAAGUUUUGAUdTdT-3=; and siSP1 (2), sense, 5=-CAUAAGCAAAG AAAUGACCdTdT-3=, and antisense, 5=-GGUCAUUUCUUUGCUUAU GdTdT-3=.

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Construction of Sp1 mutants. The Sp1 cDNA was PCR amplified from cDNA isolated from the NP460-hTERT cell line (immortalized nasopharyngeal epithelial cells) using the primers 5=-ATTAGAATTCATGG ACTACAAAGACGATGACGATAAAATGAGCGACCAAGATCA-3= and 5=-ATGCTCTAGATCAGAAGCCATTGCCACT-3=. The Sp1 PCR product containing an N-terminal FLAG tag was cloned into pcDNA3 through EcoRI and XbaI sites. Point mutations of Sp1 at the serine-101 residue were generated by PCR using the following primers: S101A forward primer, 5=-GCCATTGGACCCTGTGCAAGTTGTGTGGCTGTG3=, and S101A reverse primer, 5=-CACAGCCACACAACTTGCACAGGG TGCCAATGGC-3=; S101E forward primer, 5=-CCAGCCATTGGCACC CTGTTCAAGTTGTGTGGCTGTG-3=, and S101E reverse primer, 5=-CA CAGCCACACAACTTGAACAGGGTGCCAATGGCTGG-3=. All the sequences of the Sp1 wild type and mutants were confirmed by Sanger DNA sequencing (BGI Tech, Hong Kong). Antibodies for EBV and DDR proteins. The rabbit polyclonal BALF2, BALF5, BBLF2/3, BSLF1, and BBLF4 antibodies were provided by one of the authors (Tatsuya Tsurumi). The specificities of these antibodies have been well characterized in previous studies (13, 29). An antibody against EA-D was obtained from Jaap Middeldorp (VU University Medical Center, Netherlands). Antibodies against Zta and Rta were purchased from Argene (bioMérieux, SA). The anti-ATM serine-1981 phosphorylation rabbit polyclonal antibody and the anti-MDC1 (MDC1-50) mouse monoclonal antibody were purchased from Epitomics (Burlingame, CA) and Abcam (Cambridge, MA), respectively. The anti-␥H2AX serine-139 antibody was purchased from EMD Millipore (Massachusetts, USA). The anti-KAP1 and anti-KAP1 Ser852 phosphorylation antibodies were purchased from BD Biosciences (California, USA) and Bethyl Laboratories Inc. (Montgomery, TX, US), respectively. The RNF8 (B01P) antibody was purchased from Abnova (Taipei, Taiwan). The anti-bromodeoxyuridine (BrdU) antibody was purchased from Dako Inc. (California, USA). The anti-Sp1 (PEP-2) antibody and anti-Sp1 serine-101 phosphorylation antibody were purchased from Santa Cruz (California, USA) and Active Motif (California, USA), respectively. All the Alexa Fluor-conjugated secondary antibodies were purchased from Molecular Probes (New York, USA). Protein extraction and Western blotting. Cells were harvested by trypsinization, washed once with phosphate-buffered saline (PBS) buffer, and lysed with NETN buffer (20 mM Tris buffer, pH 8.0, 100 mM NaCl, 2 mM EDTA, 0.5% NP-40, 50 mM NaF, 2 mM sodium orthovanadate, 10 ␮M phenylmethylsulfonyl fluoride [PMSF], 2 ␮g/ml leupeptin, and 2 ␮g/ml aprotinin) on ice for 30 min. The lysates were centrifuged at 13,000 rpm for 30 min at 4°C. The supernatant was collected, and the protein concentration was determined by RC DC Protein Assay (Bio-Rad). Cell extracts were boiled in SDS sample buffer for 15 min before being analyzed by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes, and Western blotting was performed with the antibodies specified. Graded salt extraction. Equal numbers of harvested cells were subdivided into portions and lysed with NETN buffer with increasing salt contents (variable amounts of NaCl, 1 mM EDTA, 50 mM Tris-Cl, pH 8.0, 0.5% Triton X-100) for 15 min on ice. The cell lysates were then centrifuged at 2,000 ⫻ g for 15 min at 4°C, and the supernatants were collected for SDS-PAGE. Coimmunoprecipitation. Cells were harvested and lysed in cold NETN buffer supplemented with protease and phosphatase inhibitors (20 mM Tris buffer, pH 8.0, 120 mM NaCl, 2 mM EDTA, 0.5% NP-40, 50 mM NaF, 2 mM sodium orthovanadate, 10 ␮M PMSF, 2 ␮g/ml leupeptin, and 2 ␮g/ml aprotinin) for 30 min on ice. The cell lysate was then centrifuged at 14,000 rpm at 4°C for 15 min. The cell extract was collected and incubated with 1 to 2 ␮g of primary antibodies at 4°C for 4 h with constant rocking. Thirty microliters of protein A or protein G Sepharose beads (GE Amersham) was added to the cell extract and incubated for 2 h at 4°C. The beads were then washed with NETN buffer three times before incubating them with sample buffer for SDS-PAGE.

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Immunofluorescence analysis. Cells were fixed in 4% paraformaldehyde in PBS for 15 min. After washing two times with PBS, the cells were permeabilized with blocking buffer (10% FBS, 0.2% Triton X-100 in PBS) for 30 min. The cells were incubated with primary antibodies for 2 h and then washed with PBS. The cells were then incubated with secondary antibodies (Alexa Fluor 488 and 555 dye-conjugated secondary antibodies at 1:500) for 1 h before counterstaining the nucleus with Hoechst 33258 (1:5,000). For pulsed BrdU labeling, cells were incubated with 10 ␮M BrdU for 1 h. The cell culture medium was then removed, and the cells were washed twice with ice-cold PBS, preextracted with 0.5% Triton X-100 buffer for 2 min, and washed twice with PBS before fixation with ice-cold methanol for 30 min on ice. The cells were then incubated in 2 M hydrochloric acid solution containing 0.5% Triton X-100 for 10 min to denature the DNA. After removal of the hydrochloric acid and washing with PBS two times, the cells were further incubated in 0.1 M sodium borate (pH 9.0) for 5 min. The cells were then washed with PBS and blocked with blocking buffer (10% FBS, 0.2% Triton X-100 in PBS) for 30 min, followed by the steps described above. Fluorescent images were obtained using an LSM700 (Carl Zeiss, Germany) laser scanning confocal microscope with a Plan Apochromat 63⫻ 1.4-numerical-aperture (NA) oil immersion objective and equipped with a charge-coupled-device (CCD) camera using appropriate excitation and emission wavelengths. The acquired images were processed with ZEN 2011 software (Carl Zeiss, Germany). Fluorescence in situ hybridization (FISH) for the EBV genome. After BrdU staining as described above, the slide was washed twice with 1⫻ ice-cold PBS for 10 min, fixed with 4% paraformaldehyde for 10 min at room temperature, and then washed again twice with ice-cold 1⫻ PBS for 10 min each time on ice, followed by dehydration with 70%, 80%, and 95% ethanol for 2 min each at room temperature. The slide was air dried with nitrogen gas. Fifty microliters of denaturing solution (Cytocell) was added to the cell area on the slide. A coverslip was placed on the denaturing solution and sealed with rubber cement, and the slide was incubated at room temperature for 30 min. For hybridization with biotin-labeled probe, the sealed slide was first placed onto a slide moat for 4 min at 80°C. Then, the coverslip was carefully removed. The slide was then dehydrated in 70%, 80%, and 95% ethanol for 2 min each at room temperature and air dried with nitrogen gas. Three microliters of biotin-labeled EBV probe (kindly provided by Bill Sugden, McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, Madison, WI, USA) was denatured at 80°C for 6 min and incubated at 37°C for 30 min. The denatured EBV probe was added to the air-dried slide, covered with a coverslip sealed with rubber cement, and incubated in a humid box at 37°C overnight. The coverslip was then removed, and the slide was washed with 50% formaldehyde-2⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 5 min twice at 45°C and 2⫻ SSC for 5 min twice at 45°C. Thirty microliters of streptavidin-red fluorophore was pipetted onto the slide, which was then covered with a coverslip and incubated for 30 min at room temperature. After removal of the coverslip, the slide was washed with 2⫻ SSC for 5 min twice at 45°C; dehydrated with 70%, 80%, and 95% ethanol for 2 min each at room temperature; and air dried. Eight microliters of antifade DAPI (4=,6-diamidino-2-phenylindole) was added to the slide. A coverslip was placed on the slide, which was then viewed under a fluorescence microscope. Determination of infectious EBV particles produced upon lytic reactivation. A plasmid carrying the BZLF1 gene (2 ␮g) was transfected into HONE1-EBV cells to induce the lytic cycle. After triggering lytic induction, the culture medium of the EBV-infected cells was collected 48 h postinduction. The virus-containing medium was filtered through a 0.45-␮m cellulose acetate filter, and 3 ⫻ 104 Daudi cells were incubated with filtered virus-containing medium (in a 12-well cell culture plate) for 24 h. The EBV used was tagged with an EGFP gene, and infected cells could be visualized by flow cytometry analysis using a fluorescence-activated cell sorter (FACS) analyzer (CantoII; Becton Dickinson).

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Determination of the EBV genome copy number. Cells at 80% confluence were harvested by trypsinization. The cells were washed one time with PBS, and the genomic DNA was extracted using a DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s protocol. Quantitative real-time PCR (RT-PCR) of all DNA samples containing the EBV genome was performed using a LightCycler 480 Probe Master with the Universal Probe Library Set, Human (Roche Applied Science). Specific forward and reverse primers were designed with the assistance of ProbeFinder software (Roche Applied Science) as follows: EBNA1 forward primer, 5=-CC GGCTTGGTTAGTCTGTTG-3=, and EBNA1 reverse primer, 5=-AGCTTAT GCAGCACCTCAGC-3=; beta-globin forward primer, 5-GGCCCTTTTGCT AATCATGT-3=, and beta-globin reverse primer, 5=-CACACAGACCAGCA CGTTG-3=. The probes for EBNA1 and beta-globin detection are probes number 48 and number 17, respectively (Universal ProbeLibrary Set, Human; Roche). The iQ5 Real-Time PCR detection system (Bio-Rad) was used for RT-PCR experiments. Each RT-PCR was conducted under the manufacturer’s suggested conditions: a preincubation step at 95°C for 10 min and 50 cycles of amplification performed with denaturing DNA at 95°C for 10 s, followed by annealing and elongation steps at 60°C for 30 s. The concentration of DNA template was calculated based on the cycle threshold (CT) obtained from the fluorescent signal released from the PCR. RT-PCR of each sample was performed in triplicate. The RT-PCR results for each sample were normalized with the beta-globin gene, an internal control, and the CT value of each gene was set differently, as the sensitivities of the primers and probes of different EBV genes might differ from each other. The RT-PCR results were analyzed with Bio-Rad real-time PCR (BioRad) software, and the relative value of each sample was determined by the 2⫺⌬⌬CT method.

RESULTS

Lytic reactivation of EBV-infected cells elicits a DNA damage response. EBV-infected cells can be induced to undergo a productive lytic cycle by various chemical inducers, including phorbol ester (TPA), 5-azacytidine, and histone deacetylase inhibitors (HDACi), and by cross-linking the surface receptors for IgG in infected B cells using anti-IgG antibody (33, 36–38). Multiple studies have revealed the complexity of gene regulation in infected cells involved in lytic reactivation of EBV. Induction of lytic EBV infection by chemicals is cell context dependent, and the extents of lytic cycle induction by these treatments also vary in different cell types. In this report, we examine the events underlying reactivation of the EBV lytic infection in the context of nasopharyngeal epithelial cells. An EGFP-tagged EBV (strain Akata)-infected epithelial cell line model (HONE1-EBV) was used in the study. The application of TPA or sodium butyrate alone could trigger a low level of lytic infection (Fig. 1A). In combination, TPA and sodium butyrate efficiently induced lytic reactivation of EBV in infected HONE1-EBV cells. Western blot analysis showed that the expression of immediate-early proteins (Zta and Rta) and the early lytic protein EA-D of EBV was efficiently induced in these cells upon treatment (Fig. 1B). Induction of lytic reactivation was associated with efficient phosphorylation of ATM at the serine-1981 residue. Both KAP1 and histone H2AX, well-known substrates of ATM, were phosphorylated, indicating effective activation of ATM (39– 42) upon lytic reactivation of EBV infection in HONE1-EBV cells (Fig. 1B). Phosphorylation of ATM in HONE1-EBV cells was also observed in lytic reactivation induced by overexpressing the EBVborne BZLF1 gene (Fig. 1C, left). The activation of ATM after BZLF1 expression in HONE1-EBV cells was further confirmed by immunocytochemistry using the phosphorylated ATM (phospho-ATM) (S1981)-specific antibody (Fig. 1C, right). These re-


sults demonstrated the involvement of the ATM signaling pathway during lytic reactivation of EBV in infected HONE1 cells. Localization of DNA damage response proteins at the viral replication compartment during lytic EBV reactivation. Previous studies have also reported activation of the ATM signaling pathway in EBV-infected cells upon induction of lytic reactivation (18, 29–31). During lytic cycle induction in EBV-infected B cells, the ATM and MRN subunits were shown to be localized at the viral replication compartment (29). We examined if DDR proteins also localized at the viral replication compartment in HONE1-EBV cells during lytic reactivation. First, we pulse-labeled the HONE1-EBV cells induced to undergo lytic reactivation with BrdU to label cells undergoing viral DNA replication. Punctate BrdU foci, representing DNA replication sites, were observed in cell nuclei of HONE1-EBV cells upon induction of lytic infection (Fig. 2A). We validated that these BrdU foci were indeed regions of the viral replication compartment by performing an EBV-FISH study using an EBV-specific DNA probe that hybridizes to the EBV genome in infected cells. The EBV-FISH signals coincided completely with the punctate BrdU foci in HONE1EBV cells undergoing lytic reactivation, confirming that they are indeed compartmental sites of EBV replication (Fig. 2B). Localization of other replication proteins at the replication compartments in HONE1-EBV cells under lytic reactivation was also confirmed (Fig. 2C). Furthermore, phosphorylated ATM localized to these replication compartments during lytic reactivation of EBV infection. These results showed that upon induction of lytic replication in HONE1-EBV cells, both viral lytic replication proteins and phosphorylated ATM are recruited to the structural components of viral replication compartments (Fig. 2D). It has been suggested that the EBV genomes are assimilated into chromatin with a typical nucleosomal pattern (43). By using anti-␥H2AX and anti-BALF2 antibodies, we showed that ␥H2AX also colocalized with BALF2 foci at the viral replication compartment (Fig. 2D). In addition, we also observed that other DDR proteins, including phospho-ATM, MDC1, phospho-KAP1, and RNF8, were all faithfully colocalized with the viral replication proteins (Fig. 2D). These results strongly suggest the involvement of ATM activation and its downstream signaling events in the formation of the viral replication compartment in EBV-infected epithelial cells during lytic reactivation. Inhibition of ATM activity suppressed lytic EBV reactivation. We then examined if ATM activation is required for lytic viral reactivation in EBV-infected nasopharyngeal epithelial cells. Controversial findings have been reported in the literature (18, 29, 30). Earlier work suggested that ATM activation may be a side product associated with viral DNA replication (29). However, other reports support an essential role of ATM activation in viral reactivation (18, 30). The involvement of ATM in lytic reactivation of HONE1-EBV cells was examined with a specific ATM inhibitor, KU-55933 (44). KU-55933 treatment (at 10 ␮M) efficiently suppressed production of infectious EBV virions from HONE1-EBV cells stimulated by TPA and sodium butyrate to undergo lytic reactivation (Fig. 3A). Significant suppression of Zta and other early lytic proteins (Rta and EA-D) in HONE1-EBV cells was also observed. Similarly, silencing of ATM expression by siRNA also reduced the production of infectious EBV virions, similar to the result of chemically induced lytic reactivation of EBV. However, the effects of silencing of ATM by siRNA on the expression of lytic EBV protein

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FIG 1 EBV reactivation induces a DNA damage response in EBV-infected epithelial cells. (A) Recombinant HONE1-EBV green fluorescent protein (GFP) cells were treated with TPA (40 ng/ml) and sodium butyrate (3 mM) to induce the lytic cycle. The cells were observed 48 h posttreatment. The increase in the intensity of GFP fluorescence corresponds to increasing viral genome copy numbers inside the cells. (B) An ATM-dependent DNA damage response is induced during EBV reactivation by chemical inducers. HONE1-EBV cells were either mock treated or treated with TPA or sodium butyrate, alone or together, for 48 h. Cell lysates were prepared, separated by SDS-PAGE, and immunoblotted with the indicated antibodies. p-ATM, phospho-ATM. (C) (Left) BZLF1 overexpression triggers EBV reactivation and concomitantly induces a DNA damage response. BZLF1 plasmid was transfected into the cells to induce the lytic cycle. Cells were harvested at the indicated time points, and cell lysates were prepared for immunoblotting with the indicated antibodies. (Right) Cells on coverslips were transfected with BZLF1 plasmid to induce the lytic cycle. The fixed cells were immunostained with phospho-ATM (Ser1981) antibody (1:500) and counterstained with Hoechst 33258 to locate the nucleus.

were less significant than the use of a chemical inhibitor of ATM, KU-55953. Nevertheless, significant suppression of Rta was still observed. Only modest suppression of Zta and EA-D occurred in siATM-transfected HONE1-EBV cells induced to lytic reactivation (Fig. 3B). This may reflect the incomplete silencing of ATM by the siRNA approach compared to the use of a chemical inhibitor. However, effective suppression of viral DNA replication and production of infectious virions was observed in the presence of expression of Zta and EA-D. Similarly, there were also no significant alterations in the expression of early lytic EBV proteins after effective silencing of ATM expression in BZLF1-induced lytic reactivation (Fig. 3C,

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left). Lytic EBV reactivation was still significantly compromised in ATM knockdown cells, as indicated by the reduction of virion production and the decrease of EBV genome copy numbers in HONE1-EBV cells (Fig. 3C, right). These observations may suggest that the effects of ATM on lytic reactivation of EBV may not be entirely dependent on mediating the expression of early lytic viral proteins, and additional events, other than enhanced transcription of early lytic EBV protein expression, are involved in mediating the action of ATM in lytic reactivation of EBV in HONE1-EBV cells. Sp1 localization at the replication compartment is essential for efficient viral production. We then examined other signaling

Journal of Virology



FIG 2 Viral core replication proteins and DNA damage response proteins localize at the replication compartment during EBV reactivation. (A) BZLF1 plasmid was transfected into recombinant HONE1-EBV GFP cells to induce the lytic cycle. The cells were pulse-labeled with BrdU (10 ␮M) for 1 h before fixing with ice-cold pure methanol and then immunostained with anti-BrdU antibody (see Materials and Methods for details). (B) Cells were pulsed-labeled with BrdU for 1 h before fixing with methanol, and BrdU staining was performed. The cells were further fixed in 4% paraformaldehyde, and a FISH experiment was performed (see Materials and Methods for details). (C) Cells were transfected with BZLF1 plasmids. The cells were pulse-labeled with BrdU (10 ␮M) and 1 h later were fixed with pure methanol. BrdU staining was performed first, and the cells were further stained with the antibodies indicated and counterstained with Hoechst 33258 to locate the nuclei. (D) The lytic cycle was induced in cells by overexpressing BZLF1 before fixing the cells with methanol. The cells were immunostained with the antibodies indicated and counterstained with Hoechst 33258.


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FIG 3 Inhibition of ATM-dependent DNA damage signaling suppresses EBV reactivation. (A) ATM inhibitor (KU-55933) inhibits EBV reactivation in EBV-infected epithelial cells. (Top) The cells were treated with TPA (40 ng/ml) and sodium butyrate (3 mM) to induce the lytic cycle. The cells were then either mock treated or treated with KU-55933 (10 ␮M) to inhibit ATM activation. Cells were harvested at 48 h posttreatment, and cell lysates were prepared for immunoblotting with the antibodies indicated. (Bottom) Culture medium was collected and filtered through a 0.45-␮m syringe filter. The filtered medium, which contained EBV particles, was used to infect Daudi cells. The infected Daudi cells (GFP positive) were quantified by flow cytometry. (B) Knockdown of ATM suppresses EBV reactivation in EBV-infected epithelial cells. (Top) The cells were transfected with either nontargeting siRNA or siRNA targeting ATM on the first day. Another round of siRNA transfection was performed the next day, and the cells were treated with TPA (40 ng/ml) and sodium butyrate (3 mM) to induce the lytic cycle. Cells were harvested 48 h posttreatment, and cell lysate was prepared and immunoblotted with the antibodies indicated. (Bottom) Culture medium was collected and prepared as for panel A. Virion production was determined by EBV infection assay. (C) ATM knockdown suppress virion production in cells with ectopic BZLF1 expression. (Left) Cells were transfected with either nontargeting siRNA or siRNA targeting ATM on the first day and then transfected with BZLF1 plasmid to induce the lytic cycle. Cells were harvested 24 h posttransfection, and cell lysate was prepared for SDS-PAGE. Western blots were performed with the antibodies indicated. (Right) Culture medium was collected and prepared as for panel A. (Top) Virion production was determined by EBV infection assay. (Bottom) A portion of the cells were used to extract genomic DNA for EBV genome copy analysis (see Materials and Methods for details). Differences were analyzed by a paired t test (***, P ⬍ 0.001). The error bars indicate standard deviations.

pathways downstream of ATM that may be involved in EBV reactivation. Sp1 is a major signaling pathway downstream of ATM. Its involvement in lytic replication of EBV in infected B cells had been reported in earlier studies (17, 45). The involvement of Sp1 in the ATM-mediated lytic reactivation of EBV infection in epithelial cells has not been investigated. Using the yeast one-hybrid assay, the oriLyt DNA sequence harboring the downstream essential element (DEE) was shown to interact with the transcription factor Sp1 (17, 45). The DEE is one of the two DNA binding motifs (upstream essential element [UEE] and DEE) present in the cisacting element of the oriLyt of EBV important for lytic replication (7, 46). The UEE contains binding sites for Zta, while the DEE is known to be the binding site for cellular proteins, including Sp1

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(7, 46). Importantly, Sp1 was also shown to regulate the expression of EBV lytic genes, including the BZLF1 and BRLF1 genes (47–50). Sp1 has been reported to be a direct substrate of ATM upon DNA damage (51–53). Furthermore, Sp1 could be hyperphosphorylated by ATM during herpes simplex virus 1 (HSV-1) infection (54). We proceeded to examine the involvement of Sp1 in the lytic reactivation of EBV in HONE1-EBV cells. We first investigated if Sp1 is localized at the replication compartment during lytic induction. Immunocytochemistry was performed using anti-Sp1 antibody in combination with an antiBrdU and/or anti-Zta antibody to examine the localization of Sp1 in EBV-infected cells undergoing lytic reactivation. Sp1 was shown to colocalize faithfully with the foci of BrdU and Zta in

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FIG 4 Sp1 localizes and interacts with viral replication proteins at the replication compartment and is essential for efficient viral production. (A) Sp1 localizes at the viral replication compartment during the lytic cycle. HONE1-EBV cells were induced to undergo the lytic cycle by overexpressing the BZLF1 gene. Cells were preextracted and then fixed with ice-cold methanol before immunostaining. For BrdU staining, cells were first fixed in pure methanol, and the genomic DNA was denatured. The cells were immunostained with anti-Sp1, -BZLF1, and -BrdU antibodies (see Materials and Methods for details). (B) HONE1-EBV cells were transfected with FLAG-tagged BZLF1 plasmid to induce the lytic cycle. At 48 h posttransfection, cells were harvested, and total cell lysate was extracted. One microgram of the indicated antibodies was incubated with 500 ␮g of cell lysate for 2 h with constant rocking at 4°C. Protein A Sepharose beads (GE Amersham) were further incubated with the cell lysate for 1 h at 4°C. The beads were pulled down by brief centrifugation and washed three times with NETN buffer, followed by boiling in sample buffer for 10 min. The samples were separated by SDS-PAGE and immunoblotted with the indicated antibodies. IP, immunoprecipitation; WB, Western blotting. (C) Sp1 knockdown suppresses EBV virion production. (Top) Cells were transfected with SP1 siRNA [either siSP1 (1) or siSP1 (2)] on the first day of the experiment and further transfected with BZLF1 plasmid to induce the lytic cycle. Cells were harvested at 48 h posttransfection, and cell lysates were collected for Western blot analysis with the antibodies indicated. (Bottom) An EBV infection assay was performed to investigate virion production. The error bars indicate standard deviations.

HONE1-EBV cells induced to undergo lytic reactivation (Fig. 4A). To examine further if Sp1 interacts with lytic EBV proteins, we performed coimmunoprecipitation with anti-Sp1 antibody in BZLF1-overexpressing HONE1-EBV cells. As shown in Fig. 4B, the Zta (FLAG-tagged) and BALF2 proteins could be coimmunoprecipitated with Sp1. Moreover, reciprocal immunoprecipitation using anti-FLAG antibody to pull down Zta or anti-BALF2 antibody also confirmed the interaction of Sp1 with these lytic viral proteins. Collectively, our findings suggest that Sp1 interacts with viral replication proteins during viral DNA replication at the replication compartment. To further confirm the involvement of Sp1 in the regulation of EBV lytic replication, we examined if knocking down Sp1 expression with siRNA might impair lytic EBV reactivation in HONE1-EBV cells induced by BZLF1. Sp1 expression in HONE1-EBV cells was significantly knocked down by siRNA


transfection, as confirmed by Western blotting analyses (Fig. 4C, top). Interestingly, the expression of early lytic proteins was not significantly affected despite effective knockdown of Sp1 in HONE1-EBV cells. However, knocking down Sp1 in HONE1-EBV cells significantly inhibited viral DNA replication and production of infectious EBV particles (Fig. 4C, bottom). Collectively, these results support an interactive role of Sp1 with EBV lytic proteins in the replication of viral DNA and virion production. Similar to the observation of silencing ATM, additional events, other than suppression of expression of lytic viral proteins, appear to be involved. ATM mediates Sp1 phosphorylation at serine-101 during EBV reactivation. We next examined if phosphorylation of Sp1, which is a downstream event of ATM activation, might be involved in lytic reactivation of EBV infection in HONE1-EBV cells. The phosphorylation status of Sp1 during lytic cycle induction

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FIG 5 Sp1 is phosphorylated at serine-101 by ATM during EBV reactivation. (A) Individual clones of recombinant HONE1-EBV GFP cells were isolated with cloning cylinders. The clones were separately tested for lytic cycle induction by overexpressing the BZLF1 gene, and clones 1 and 2 were selected to perform the subsequent experiments. Cells were harvested at 36 h posttransfection, and cell lysate was prepared for SDS-PAGE. The arrows indicate the migration of protein bands of Sp1 after induction of the lytic cycle. (B) Clone 1 and clone 2 cells were subjected to transient overexpression of the BZLF1 gene to induce the lytic cycle. At 8 h posttransfection, the cells were either mock treated or treated with 10 ␮M KU-55933 for 24 h. Cells were harvested, and cell lysate was prepared for SDS-PAGE. Immunoblotting was performed with the antibodies indicated. (C) The phosphodefective Sp1 mutant cannot localize at the replication compartment during lytic induction. (Bottom left) FLAG-tagged Sp1 mutants were created by PCR mutagenesis using wild-type (WT) FLAG-tagged Sp1 as the template. S101A and S101E are the phosphodefective and phosphomimetic Sp1 mutants, respectively. The expression of the FLAG-Sp1 wild type and mutants was tested by overexpressing the genes in HONE1-EBV clone 2 cells. Cell lysate was collected for SDS-PAGE analysis and immunoblotted with the antibodies indicated. (Top) BZLF1 plasmids, together with either the FLAG-Sp1 wild type or mutants, were cotransfected into HONE1-EBV clone 2 cells. At 48 h posttransfection, the cells were fixed in methanol, followed by immunostaining with anti-BALF2 and anti-FLAG (M2) antibody. The cells were counterstained with Hoechst 33258 to locate the nuclei. (Bottom right) Summary of the results of colocalization of the Sp1 wild type or mutants at the replication compartment during lytic induction. More than 100 BALF2 and FLAG double-positive cells were counted, and the experiment was performed in triplicate. Differences were analyzed by a paired t test. **, P ⬍ 0.005. The error bars indicate standard deviations.

was examined in two homogeneous populations of HONE1-EBV cells selected for enhanced responsiveness toward lytic induction (clones 1 and 2) to dissect the molecular details of Sp1-mediated lytic reactivation. As shown in Fig. 5A, Sp1 extracted from control cells before induction of lytic reactivation migrates as two major species in SDS-PAGE, representing the phosphorylated and unphosphorylated Sp1 isoforms. In contrast, expression of BZLF1 in EBV-infected cells resulted in a shift of isoforms of Sp1 to the upper band (the phosphorylated form). We then carried out further investigations to examine the phosphorylation status of Sp1 using specific antibody against the phosphorylated residue of Sp1. Previous reports showed that Sp1

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possesses 15 putative target sites (SQ/TQ) that are spread between the two SQ/TQ cluster domains (51, 54). Among them, serine-56 and serine-101 were reported to be associated with ATM-dependent phosphorylation during the DNA damage response or HSV-1 infection. Furthermore, only the serine-101 residue was found to be phosphorylated by ATM in vivo and in vitro (54). Using a specific antibody, we confirmed, by Western blotting, an increase in phosphorylation of Sp1 at the serine-101 residue during induction of lytic reactivation (Fig. 5B). Treatment of cells with the ATM inhibitor KU-55933 abolished the increase in serine-101 phosphorylation during lytic reactivation of EBV infection, indicating that this specific Sp1 phosphorylation site is dependent on ATM activation.

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The role of serine-101 phosphorylation in localization of Sp1 at replication compartments. To further investigate the functional significance of Sp1 serine-101 phosphorylation during lytic infection, we conducted mutation analysis to study if Sp1 serine101 phosphorylation is required for its localization at the replication compartment. We constructed FLAG-tagged Sp1 mutants by replacing the serine-101 residue with either alanine (S101A) or glutamic acid (S101E). The S101A Sp1 mutant mimics the phosphodefective Sp1 at the serine-101 residue, whereas the S101E mutant is the phosphomimetic form of Sp1 (Fig. 5C, lower left). We transfected individual FLAG-tagged Sp1 (wild type or mutants) into HONE1-EBV cells induced to undergo lytic reactivation of EBV by expressing the BZLF1 gene. The localization of the FLAG-tagged Sp1 proteins during induction of viral replication was examined (Fig. 5C, top). Consistent with our earlier observations, more than 80% of the wild-type FLAG-Sp1 foci colocalized with BALF2 at the replication compartment. In addition, a comparable amount of the S101E phosphomimetic mutant also colocalized faithfully with BALF2 foci at replication compartments. In contrast, only 20% of the S101A phosphodefective mutant cells were found to colocalize with the BALF2 foci (Fig. 5C, lower right). These results support the role of phosphorylation of Sp1 at the serine-101 residue for its localization at replication compartments during lytic reactivation of EBV in infected HONE1 cells. Sp1 is involved in the localization of viral replication proteins to the replication compartment. Having confirmed that phosphorylated Sp1 (serine-101) localizes at the replication compartment upon induction of lytic infection in EBV-infected HONE1 cells, we sought to examine if Sp1 might be involved in the localization of viral replication proteins to replication compartments during lytic reactivation. To address this, we examined the localization of several EBV lytic replication proteins to the replication compartment using an immunocytochemistry method in HONE1-EBV cells after knocking down Sp1 expression with siRNA. The localization of these EBV lytic replication proteins upon lytic reactivation in both control cells and Sp1 knockdown cells was revealed by specific antibodies against EBV lytic replication proteins (Fig. 6A). Strikingly, the formation of EA-D, Zta, and BALF2 foci in Sp1-depleted cells was remarkably impaired. Furthermore, formation of viral replication foci of BBLF4 and BBLF5 replication proteins was also suppressed in Sp1 knockdown cells (Fig. 6B). The recruitment of both host and viral proteins to the replication compartment during lytic reactivation of EBV infection in HONE1-EBV cells suggested functional interaction of these proteins with the viral chromatin structure. We then examined if Sp1 may facilitate the binding of these viral replication proteins to chromatin by examination of their extractability by graded concentrations of salt solution. As shown in Fig. 6C, we observed increasing amounts of viral proteins accumulated in high-salt fractions, suggesting their close association with viral DNA during lytic cycle induction. In contrast, a decreased association of most of the viral replication proteins (BALF2, BALF5, BSLF1, EA-D, and Zta [FLAG]) was observed in Sp1 knockdown cells, which supports a role of Sp1 in facilitating interaction of viral lytic proteins with the viral chromatin. The detailed mechanisms involved remain to be elucidated. Sp1 serine-101 phosphorylation is involved in the recruitment of viral replication proteins to the replication compartment. To further confirm involvement of Sp1 serine-101 phos-


phorylation in the lytic replication of EBV DNA, we examined the ability of a phosphomimetic Sp1 mutant to restore the recruitment of viral replication proteins to the replication compartment in Sp1 knockdown cells. Sp1 knockdown suppressed formation of the replication compartment of EBV during lytic reactivation, as shown by the reduction of cells with punctate BrdU foci in the Sp1 knockdown cells (Fig. 7A). The viral BALF2 was revealed as punctate foci during lytic DNA replication. Costaining of BALF2 and BrdU was used to examine if Sp1 knockdown cells could suppress viral DNA replication. Sp1 knockdown resulted in a decrease in colocalization of BALF2 with BrdU foci after induction of lytic reactivation (Fig. 7B). Transient reconstitution of the wild type and the S101E phosphomimetic mutant rescued the knockdown phenotype (Fig. 7C). Conversely, overexpression of the S101A Sp1 mutant could not rescue the knockdown phenotype. We also examined the EBV genome copy number in cells reconstituted with different Sp1 constructs. Expression of the wild type and the phosphomimetic mutant (S101E) resulted in a 2- to 3-fold increase in the EBV genome copy number, while the phosphodefective mutant (S101A) could enhance the EBV genome copy number by only 0.5-fold (Fig. 7D). These results support the role of Sp1 serine-101 phosphorylation in the recruitment or stabilization of replication proteins at the replication compartment essential for replication of EBV DNA. The Sp1 phosphomimetic mutant (S101E) rescued the defect of viral DNA replication in ATM knockdown cells. As ATM knockdown cells were deficient in undergoing viral DNA replication, we further examined if the Sp1 phosphorylation at the serine-101 residue could rescue the replication defect of ATM knockdown cells. Knockdown of ATM suppressed lytic EBV replication, as shown by the reduction of cells with punctate BrdU foci (Fig. 8A). We then examined the effects of different Sp1 mutants on rescuing lytic reactivation in ATM knockdown cells. We counted the cells that exhibited costaining of BrdU and BALF2 (Fig. 8B). The Sp1 S101E phosphomimetic mutant could partially restore viral DNA replication in some cells, but not the S101A defective mutant (Fig. 8C). By real-time PCR, the EBV genome copy number also increased after reconstitution with the phosphomimetic Sp1 mutant (Fig. 8D). To eliminate the possibility that the defect in viral DNA replication in ATM or Sp1 knockdown cells was not due to a decrease in viral core lytic replication protein expression, we examined the protein levels of those EBV core replication proteins in Sp1 or ATM knockdown cells. Overexpression of the BZLF1 gene in control HONE1-EBV cells could induce lytic reactivation, as revealed by the induced expression of six core replication proteins (excluding Zta, as it was overexpressed by a transfected BZLF1-expressing plasmid). In ATM or Sp1 knockdown cells, the expression of six core replication proteins (EA-D, BBLF4, BBLF2/3, BSLF1, BALF2, and BALF5) could still be detected at levels comparable to those in the control cells (Fig. 8E). Therefore, the defect of lytic reactivation in ATM and Sp1 knockdown cells might not be related to a decrease in the expression levels of these viral replication proteins but dependent on the localization at the replication compartments. In summary, we have investigated and confirmed the active involvement of ATM activation in lytic reactivation of EBV in infected nasopharyngeal epithelial cells. Our results suggest that Sp1 phosphorylation at the serine-101 residue, one of the downstream events of ATM activation, plays an important role in facil-

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FIG 6 Sp1 knockdown leads to failure of viral replication proteins to localize at the replication compartment. (A) HONE1-EBV cells were transfected with either control siRNA or SP1 siRNA before inducing the lytic cycle by overexpressing BZLF1. At 48 h posttransfection, the cells were preextracted, fixed with methanol, and immunostained with the indicated antibodies. (B) Experiments similar to those in panel A were performed, but the cells were immunostained with other viral replication proteins. (C) Control siRNA or SP1 siRNA was transfected into cells on the first day. The FLAG-BZLF1 gene was further transfected to induce the lytic cycle the next day. At 24 h posttransfection, cells were harvested, and cell lysates were extracted with NETN buffer with increasing salt contents. Western blotting was performed with the antibodies specified.

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FIG 7 Robust lytic DNA replication depends on Sp1 serine-101 phosphorylation at the replication compartment. (A) HONE1-EBV cells were transfected with either control siRNA or SP1 siRNA and were further transfected with BZLF1 plasmid to induce the lytic cycle. At 48 h posttransfection, the cells were incubated with 10 ␮M BrdU for 1 h. The cells were then preextracted, fixed with methanol for 15 min, and immunostained with the antibodies indicated (for details, see Materials and Methods). (B) Endogenous Sp1 in HONE1-EBV clone 2 cells was depleted with SP1 siRNA before cotransfecting with BZLF1 and either the wild type or the SA (S101A) or SE (S101E) Sp1 mutant. At 48 h posttransfection, the cells were incubated in culture medium containing 10 ␮M BrdU for 1 h and then preextracted before fixing with methanol for 15 min. After denaturing the DNA, the cells were immunostained with the BALF2 and BrdU antibodies and counterstained with Hoechst 33258 (for details, see Materials and Methods). The arrowheads indicate cells that expressed BALF2 protein but where no lytic DNA replication occurred. (C) Summary of the results from both control and Sp1-depleted cells after reconstitution with different Sp1 constructs. More than 200 cells were scored in each group, and the experiment was performed in triplicate. Differences were analyzed by a paired t test: **, P ⬍ 0.005. (D) Genomic DNAs of different Sp1-reconstituted cells were extracted for EBV genome copy analysis by real-time PCR. Relative EBV copy numbers are shown. Differences were analyzed by a paired t test. *, P ⬍ 0.05. The error bars indicate standard deviations.

itating the recruitment of viral lytic proteins to the replication compartment. DISCUSSION

Previous studies have reported a correlation between ATM activation and lytic reactivation of EBV (18, 29, 30, 55). DNA repair proteins, like homologous recombinational repair (HRR) and mismatch repair (MMR) factors, have been shown to localize to the replication compartment during lytic reactivation of EBV in infected B cells (32, 56). The involvement of ATM activation and DDR proteins in lytic reactivation of EBV had been reported in earlier studies (18, 29, 30, 32), but their roles in the formation of viral replication compartments in infected epithelial cells remain largely undefined. In this study, we demonstrated that ATM activation plays an essential role in the formation of viral replication compartments during lytic reactivation of EBV in the context of infected nasopharyngeal epithelial cells. An EGFP-tagged EBV (strain Akata)-infected epithelial cell


line model (HONE1-EBV) was used in this study. The low “lytic” background of HONE1-EBV cells allowed us to decipher the role of ATM activation in the formation of the replication compartment. The use of EBV-FISH and BrdU pulse-labeling allowed us to localize the site of replication of viral DNA during lytic reactivation of EBV infection in HONE1-EBV cells. We observed the localization of activated ATM and DDR signaling mediators to these discrete viral replication compartments in the nuclei of EBV-infected cells, suggesting their involvement in lytic viral replication. A proposed function of ATM activation in lytic reactivation in EBV-infected cells is repair of DNA double-strand breaks generated during lytic replication of viral DNA. During lytic reactivation, the EBV DNA is replicated in a rolling-circle manner into a linearized viral DNA and eventually cleaved into individual viral genomes for packaging into infectious virions. The cleavage of the replicated viral DNA generates double-strand DNA breaks that may activate DDR signaling (32). This proposed function of ATM

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FIG 8 Partial rescue of viral DNA replication in ATM-depleted cells by overexpressing the phosphomimetic Sp1 serine-101 mutant. (A) HONE1-EBV cells were transfected with either control siRNA or ATM siRNA before inducing the lytic cycle by overexpressing BZLF1. The cells were processed for immunostaining with the antibodies indicated. (B) Endogenous ATM in HONE1-EBV clone 2 cells was depleted with ATM siRNA before cotransfecting with BZLF1 and either the wild type or the S101A or S101E Sp1 mutant. At 48 h posttransfection, the cells were incubated in culture medium containing 10 ␮M BrdU for 1 h and then preextracted and fixed with methanol for 15 min. After denaturing the DNA, the cells were blocked with 10% FBS in PBS before immunostaining with the BALF2 and BrdU antibodies and counterstaining with Hoechst 33258 (for details, see Materials and Methods). (C) Summary of the results from both control and ATM-depleted cells after reconstitution with different Sp1 constructs. More than 200 cells were scored in each group, and the experiment was performed in triplicate. Differences were analyzed by a paired t test. *, P ⬍ 0.05. (D) Genomic DNAs of different Sp1 reconstituted cells were extracted for EBV genome copy analysis by real-time PCR. Relative EBV copy numbers are shown. Differences were analyzed by a paired t test. *, P ⬍ 0.05; **, P ⬍ 0.005. (E) (Right) HONE1-EBV clone 2 cells were transfected with either control siRNA, ATM siRNA, or SP1 siRNA for 24 h before cotransfecting with BZLF1 and different FLAG-Sp1 constructs. At 36 h posttransfection, cells were harvested, and whole-cell lysate was prepared for Western blot analysis. To check the knockdown efficiency of the siRNA transfection, protein samples of vector-transfected cells in each siRNA group were analyzed by Western blotting. Shown is the efficient knockdown of endogenous ATM and Sp1 in HONE1-EBV clone 2 cells in the experiment. The error bars indicate standard deviations.

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activation and DDR signaling was supported by reports that DNA repair factors are spatiotemporally recruited to the newly synthesized viral DNA at the replication compartments (32, 56, 57). While confirming the activation of ATM during lytic reactivation of EBV in infected nasopharyngeal carcinoma cells, we also demonstrated active participation of ATM activation in the recruitment of EBV replication proteins to the viral replication compartments essential for lytic replication of EBV DNA. Furthermore, we have also identified phosphorylation of Sp1 at the serine-101 residue as a major signaling event downstream of ATM activation contributing to the formation of viral replication compartments. The use of the specific ATM kinase inhibitor KU-55933 and knockdown of ATM expression by siRNA allow us to dissect in detail the molecular events associated with ATM activation during lytic reactivation of EBV and recruitment of viral lytic proteins to the viral replication compartment in infected nasopharyngeal carcinoma cells (Fig. 3). Inhibiting ATM activation by KU-55933 resulted in reduced production of infectious virions (Fig. 3A and B) and, interestingly, reduction of immediate-early and early lytic protein (Zta, Rta, and EA-D) levels in ATM inhibitor-treated cells (Fig. 3A). The reduction of early lytic viral proteins may be responsible for the inhibition of viral replication. These results agree with another recent study reporting that ATM kinase activity is required for lytic viral replication mediated by chemical induction and is involved in the transcription of immediate-early lytic genes in a variety of cell lines (30). The use of chemicals, including TPA and sodium butyrate, to induce lytic replication may induce DNA damage and activate ATM signaling, which may complicate the interpretation of the role of ATM in lytic viral reactivation. Induction of lytic reactivation of EBV in infected nasopharyngeal carcinoma cells was further examined by expression of BZLF1, the master EBV gene, to switch on lytic reactivation. Again, ATM activation was observed in HONE1-EBV cells expressing BZLF1. Knockdown of ATM abolishes recruitment of viral replication proteins to replication compartments, suggesting a role of ATM in the formation of the replication compartment. Inhibition of replication compartment formation by ATM siRNA was still observed in the presence of BZLF1 overexpression, indicating that other downstream events of ATM activation are involved. We have identified the involvement of Sp1 phosphorylation, a key downstream event of ATM activation, in lytic reactivation of EBV in infected epithelial cells. Earlier studies had reported that cellular transcription factors are involved in the regulation of viral lytic replication in EBV-infected cells (17). Cellular transcription factors could promote viral DNA replication by binding to the oriLyt enhancer region of EBV. Sp1 has been characterized as a bona fide transcription activator of the Z promoter and the R promoter (49, 58). Recent studies have suggested that Sp1 cooperates with protein kinase C␦ (PKC␦) and p53 to regulate Z promoter activity (49, 50). Sp1 and ZBP-89 also interact directly with the viral replication proteins EA-D and BALF5 (17). Sp1 is also involved in many cellular processes, including cell proliferation, senescence, and apoptosis (59). In this study, knockdown of endogenous Sp1 expression suppressed lytic EBV reactivation (Fig. 4C and D), supporting its role in lytic reactivation of EBV (49, 60–63). Furthermore, Sp1 localized at replication compartments during lytic reactivation (Fig. 4A) and presumably interacted with viral core replication proteins in EBV-infected epithelial cells to facilitate lytic reactivation (Fig. 4B). We next investigated in detail the role of Sp1 phosphorylation in the formation of the replication


compartment during induction of lytic EBV reactivation in the context of nasopharyngeal epithelial cells. In Sp1 knockdown cells, EBV lytic replication proteins failed to localize at replication compartments, supporting the role of Sp1 in the formation of replication compartments (Fig. 6A and B). Sp1 may also play a direct role in regulating early lytic genes required for EBV lytic infection, as evidenced by the presence of the Sp1 DNA binding element in the promoters of lytic genes (48, 50, 60, 64, 65). However, the role of Sp1 in localization of EBV replication proteins to replication compartments may not be directly related to its transcriptional activity on early viral lytic genes. Our immunoblotting analyses did not reveal major alterations in the protein levels of core viral lytic proteins in Sp1 knockdown cells (Fig. 8E). By extracting the viral replication proteins with graded salt contents, we observed that the replication proteins in Sp1 knockdown cells were more easily extracted with low-salt protein extraction buffer than those in control cells (Fig. 6C), suggesting a role of Sp1 in facilitating the binding of viral replication proteins to viral chromatin. The detailed mechanisms involved are unclear at this stage. Interestingly, a recent study reported that an ATM activator, phosphorylated TIP60, also localizes to viral lytic gene promoters to facilitate lytic gene transcription (18). It has been noted that TIP60 drives only specific lytic gene expression by binding to certain lytic gene promoters (Rta, LMP1, and BHLF1) after lytic induction in Akata (EBV) cells. While the detailed mechanisms by which TIP60 activates the transcription of lytic EBV genes remains to be defined, chromatin histone acetylation at the lytic gene promoters, which allows the relevant transcription factors access to the promoters of target genes for transcription, may be involved. The action of Sp1 to facilitate lytic replication may differ from that of TIP60. In our experimental model, we did not observe a dramatic change in replication protein expression upon Sp1 knockdown. This discrepancy may be attributed to the effect of BZLF1 overexpression, which stimulates lytic gene expression effectively. Our study showed that formation of replication compartments by localizing the replication proteins to the EBV DNA may be the prime function of Sp1 in lytic reactivation of EBV in infected epithelial cells. Sp1 is a downstream substrate of activated ATM following genotoxic stress (51, 52, 66). It has been shown that Sp1 is phosphorylated by ATM at the serine-101 residue after DNA damage (51, 66). Our findings indicate that ATM is a primary mediator of Sp1 serine-101 phosphorylation during lytic induction. In addition to ATM, ATR and DNA-PK are the other two potential kinases that phosphorylate Sp1 (67, 68). Their involvement in phosphorylation of Sp1 and effects on the formation of the EBV replication compartment were not investigated in this study and remain to be defined. The importance of phosphorylation of the serine-101 residue of Sp1 was further confirmed by Sp1 mutant analysis. Only the Sp1 phosphomimetic mutant (Sp1 S101E) and not the phosphodefective mutant (Sp1 S101A) could be localized at the viral replication compartments during lytic reactivation (Fig. 5C). The defective replication phenotype in Sp1 knockdown cells could be rescued only by reconstitution of wild-type Sp1 or the phosphomimetic Sp1 mutant (Fig. 7C and D). Additionally, we found that only the Sp1 S101E mutant could partially rescue the defect in viral DNA replication in ATM knockdown cells, suggesting that additional events are involved (Fig. 8C and D). ZBP-89 is another transcription factor that has been reported to contribute to viral

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DNA replication (17) and regulates p21 expression in response to histone deacetylase inhibitor treatment (53). Nonetheless, our results indicate that the ATM-mediated phosphorylation of Sp1 at the serine-101 residue is an important event in the lytic replication of EBV DNA. A proposed function of Sp1 in lytic viral replication is to serve as a scaffold protein to promote the binding of viral core replication proteins to the oriLyt. The ATM-mediated Sp1 phosphorylation at the serine-101 residue may provide docking sites for viral replication proteins to be tethered at replication compartments during lytic cycle induction. In conclusion, we report in this study that activation of ATM and Sp1 phosphorylation and its localization at replication compartments are important events for EBV DNA replication and the production of infectious virions. ATM phosphorylation of Sp1 at the serine-101 residue promotes retention of replication factors at the replication compartments. Our study supports the active involvement of ATM signaling in lytic replication of EBV in infected epithelial cells, which are believed to be the primary cellular sites in EBV-infected cells for lytic replication of EBV transmission. EBV hijacks host ATM signaling in infected epithelial cells to replicate viral DNA during lytic reactivation. ACKNOWLEDGMENTS We appreciate the kind gifts from Bill Sugden (University of Wisconsin— Madison, Madison, WI, USA). We also thank laboratory members for constructive criticism of the manuscript. Imaging/flow cytometry data were acquired using equipment maintained by the University of Hong Kong Li Ka Shing Faculty of Medicine Faculty Core Facility. We acknowledge the assistance of the University of Hong Kong Li Ka Shing Faculty of Medicine Faculty Core Facility. This work was supported by the following grants: Research Grants Council, General Research Fund, grants HKU 779312 and HKU 779713; Research Grants Council, Area of Excellence Scheme, grant number AoE/ M-06/08; Research Grants Council, Theme-Based Research Scheme, grant number T12-401/13-R; and a Hong Kong University CRCG grant. We declare that no competing interests exist.

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