Kaposi's Sarcoma-Associated Herpesvirus Induces ATM and H2AX. DNA Damage Response Early During de novo Infection of Primary

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1 JVI Accepts, published online ahead of print on 18 December 2013 J. Virol. doi: /jvi Copyright 2013, American Society for Microbiology. All Rights Reserved. JVI Revised Manuscript Kaposi's Sarcoma-Associated Herpesvirus Induces ATM and H2AX DNA Damage Response Early During de novo Infection of Primary Endothelial Cells Which Play Roles In Latency Establishment Vivek Vikram Singh, Dipanjan Dutta, Mairaj Ahmed Ansari, Sujoy Dutta and Bala Chandran* H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL U.S.A. Running title: KSHV induces selective DDR during de novo infection *Corresponding author. Mailing address: Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL Phone (847) ; Fax (847)

2 ABSTRACT DNA Damage Response (DDR) that evolved to repair host cell DNA damages also recognizes viral DNA entering the nucleus during infections. Here, we investigated the modulation of DDR signaling during de novo infection of primary endothelial cells by KSHV. Phosphorylation of representative DDR-associated proteins such as Ataxia Telangiectasia Mutated (ATM) and H2AX were induced as early as 30 min (0.5h) post infection which persisted during in vitro KSHV latency. Phosphorylated H2AX ( H2AX) colocalized at 30 min (0.5h) with the KSHV genome entering the nuclei. Total H2AX protein levels also increased which is attributed to a decrease in degradative H2AX Lys 48-linked polyubiquitination with concomitant increase in Lys 63-linked polyubiquitination that is shown to increase protein stability. ATM and H2AX phosphorylation and H2AX nuclear foci were also induced by UV-inactivated KSHV which ceased at later times of infection. Inhibition of ATM kinase activity by KU55933 and H2AX knockdown by sirna significantly reduced the expression of KSHV latency associated nuclear antigen (ORF 73; LANA- 1) and LANA-1 nuclear puncta. Knockdown of H2AX also resulted in >80% reduction in the nuclear KSHV DNA copy numbers. Similar results were also observed in ATM (-) cells although comparable levels of viral DNA entered the ATM (-) and ATM (+) cell nuclei. In contrast, knockdown of CHK1 and CHK2 did not affect ORF73 expression. Collectively, these results demonstrated that KSHV induces ATM and H2AX, a selective arm of the DDR, for the establishment and maintenance of its latency during de novo infection of primary endothelial cells. 2

3 IMPORTANCE Eukaryotic cells mount a DNA Damage Response (DDR) to sense and repair different types of cellular DNA damage. In addition, DDR also recognizes exogenous genetic material such as the viral DNA genome entering the nucleus during infections. The present study was undertaken to determine whether de novo Kaposi's sarcomaassociated herpesvirus (KSHV) infection modulates DDR. Our results demonstrate that early during de novo infection of primary endothelial cells, KSHV induces a selective arm of the DDR signaling such as the ATM kinase and its downstream target H2AX, which are essential for KSHV s latent gene expression and establishment of latency. These studies suggest that targeting ATM and H2AX could serve as an attractive strategy to block the establishment of KSHV latent infection and the associated malignancies. 3

4 INTRODUCTION Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), a 2-herpesvirus, is etiologically associated with Kaposi's sarcoma (KS), an angioproliferative malignancy of human skin, body cavity-based B-cell lymphoma (BCBL) (or primary effusion lymphoma; PEL), and some forms of polyclonal B-cell proliferative multicentric Castleman s Disease (MCD) (1). In vivo, viral DNA and transcripts have been detected in human B cells, macrophages, keratinocytes, endothelial cells, and epithelial cells (2). KSHV infects a variety of in vitro target cells such as human dermal microvascular endothelial cells (HMVEC-d), foreskin fibroblast cells (HFF), embryonic kidney epithelial cells (293), monocytic cells (THP- 1) and B cells. KSHV entry into target cells is mediated by endocytosis, followed by rapid transit of the viral genome-containing capsid along the microtubule network to nuclear pores and subsequent delivery of viral double strand (ds)-dna genome into the nucleus (3). As in other herpesviruses, the virion associated KSHV genome is not associated with nucleosomes, histones or any other host DNA binding proteins (4, 5). Unlike α- and β-herpesviruses, 2-KSHV primary infection of target cells in vitro does not result in a productive lytic cycle and progeny viral particle formation. Instead, the virus enters into latency with limited latent viral gene expression, and the viral genome adopts a chromatin structure similar to that of the host cell s chromosomes and persists in the host cells as a circular episome (2). Mammalian cells possess extensive regulatory signaling mechanisms such as DNA Damage Response (DDR) to sense and repair different types of cellular DNA damage (6). DDR is a signal transduction cascade and lesions in the DNA are detected by the DDR sensor proteins which activate kinases, which in turn lead to amplification of the signals through a series of downstream effector molecules. 4

5 Spearheading the DDR signaling pathways are the phosphoinositide-3-kinase (PI3)- like kinases Ataxia Telangiectasia Mutated (ATM), ATM and RAD3-related (ATR) and DNA-dependent protein kinase (DNA-PK). These Ser/Thr kinases regulate cellcycle checkpoint control, DNA replication, DNA repair, and apoptosis in response to genotoxic stress (7, 8). ATM is activated at double-stranded breaks (DSBs), while ATR responds to single stranded lesions. The Mre11-Rad50-Nbs1 (MRN) complex, considered to be the sensor for DSBs, efficiently activates ATM, which becomes autophosphorylated and phosphorylates large subsets of downstream targets that regulate cell cycle checkpoint and repair (9). Among the proteins phosphorylated in the DNA damage cascade are the mediators of repair (H2AX, BRCA1, 53BP1 and Mdc1) and effectors of the checkpoint responses (CHK1 and CHK2) that modulate the cell cycle until the repair is complete (10). One of the foremost proteins to be phosphorylated upon DNA damage is the histone variant H2AX at serine 139 (referred to as H2AX), which acts as a signal in recruitment of DNA damage proteins to DSBs and is used as a biomarker for the DNA damage response (11). In addition to cellular DNA damage, the host s repair machinery also recognizes exogenous genetic material such as the viral DNA genome entering the nucleus during infections (12-14). However, several studies show that many DNA viruses manipulate and perturb the key signaling events of DDR pathways for their own advantage (15). For example, during lytic infection in Vero or HeLa cells leading into progeny virus formation, wild-type herpes simplex virus type 1 (HSV-1; KOS; 17syn + ) induces DDR signaling by recruiting the cellular recombination and repair proteins to the sites of DNA replication (16), and by phosphorylating ATM kinase which leads into the induction of associated signaling of downstream target proteins (17) including the phosphorylation of H2AX ( H2AX) and marginalization of 5

6 H2AX to the periphery of viral replication centers (18). In contrast, although human cytomegalovirus (HCMV-strain AD169) infection of fibroblast cells induced H2AX phosphorylation, the resulting DDR was inhibited by mislocalization of checkpoint proteins (19), and H2AX colocalized with HCMV replication compartments. Activation of H2AX was dependent on viral replication as it did not occur in p53 mutant cells in which HCMV replication is abrogated (19, 20). Earlier studies have examined the effects of individual KSHV genes on the DDR. Ectopic expression of KSHV latent v-cyclin protein in telomere immortalized human endothelial cells leads to DDR activation as evidenced by the phosphorylation of ATM, H2AX, CHK2 and p53, and S-phase arrest (21). Additionally, KSHVencoded latency-associated nuclear antigen (LANA-1) had a role in H2AX phosphorylation and H2AX colocalized with the complex between LANA-1 and the Terminal Repeats (TRs) of the KSHV episome, an essential step in tethering of viral DNA to the host chromosome (22). In contrast, KSHV v-interferon regulatory factor 1 (v-irf1), an immediate early (IE) lytic protein, inhibited the ATM signaling during reactivation by direct interaction with the ATM kinase (23). Interestingly, infection with the related murine -herpesvirus 68 (MHV68) also phosphorylated H2AX which has been attributed to the activity of the viral kinase, ORF36, along with ATM (24). ATM and H2AX, as well as ORF36 were collectively shown to be essential for optimal in vivo MHV68 replication in mouse macrophages (25). Furthermore, ORF36 and H2AX were reported to facilitate the transcription of MHV68 Replication and Transcription Activator (RTA) and replication of viral DNA during de novo lytic infection of primary macrophages (26). The present study was undertaken to determine whether de novo KSHV infection modulates DDR. Results presented here demonstrate that early during de 6

7 novo infection of primary endothelial cells, KSHV induces the DDR signaling by activating the ATM kinase, which is essential for the phosphorylation of H2AX that formed distinct multiple colocalization spots ( H2AX-foci) with KSHV genome in the nuclei of the infected cells. Furthermore, these studies demonstrate that both ATM and H2AX play important roles in latent gene expression and establishment of KSHV latency. Downloaded from on October 1, 2018 by guest 7

8 MATERIALS AND METHODS Cells. Human microvascular endothelial cells of dermal origin-hmvec-d, CC-2543 (Lonza, Walkersville, Maryland) were cultured in endothelial basal medium 2 (EBM- 2) with growth factors (Lonza, Walkersville, Maryland) as described before (27-29). KSHV-positive BCBL-1 PEL cells were cultured in RPMI 1640-Glutamax (Gibco Life technologies, Grand island, NY). ATM-positive G-361 (ATCC CRL-1424 TM ) and ATM-deficient HT-144 (ATCC HTB-63 TM ) melanoma cells were purchased from ATCC and cultured in ATCC-formulated McCoy s 5a modified medium (catalog no ). RPMI 1640-Glutamax and McCoy s 5a medium were supplemented with 10% heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals Inc. Lawrenceville, GA), and PenStrep antibiotics (Gibco Life Technologies, Grand Island, NY). Virus. The induction of the KSHV lytic cycle in BCBL-1 cells, supernatant collection, and virus purification procedures were described previously (27-29). To prepare replication-defective virus, KSHV was inactivated with UV light (365 nm) for 20 min at a 10-cm distance. To generate 5-bromo-2-deoxyuridine (BrdU)-labeled KSHV virus to enable KSHV-genome labeling, a modified protocol previously described to produce BrdU-labeled HCMV (30) was utilized. Briefly, upon addition of 20ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce the lytic (productive) cycle of KSHV in BCBL-1 cells, BrdU Labeling Reagent (Life Technologies, Camarillo, CA) diluted to 1:100 (from supplied concentrate) was added to the culture medium as per the manufacturer s instructions. Flasks with media containing BrdU were kept in dim light during incubation to avoid photolysis of BrdU residues. The supernatant collection and virus purification procedures were carried out as per the previously established standard procedures in the laboratory (27-29). To 8

9 determine the KSHV titer, KSHV DNA was extracted from the purified virus, and copy numbers were quantitated by real-time DNA PCR using primers amplifying the KSHV ORF 73 gene (27-29). Antibodies and reagents. Rabbit anti-human pser139-h2ax ( H2AX), H2AX, pser296-chk1, CHK1, pthr68-chk2, CHK2, pser1524-brca1, BRCA1, pser1981-atm, ATM, pthr202/tyr204-erk1/2 and ERK2 antibodies were from Cell Signaling Technology, Beverly, MA. Mouse monoclonal antibodies against human phospho-histone H2AX (Ser 139) for IFA were from EMD Millipore, Billerica, MA. Mouse anti-polyubiquitin-k63 and rabbit anti-polyubiquitin-k48 monoclonal antibodies were from Millipore, Temecula, CA. Mouse monoclonal antibodies against -actin and -tubulin were from Sigma, St. Louis, MO. Rabbit anti- BrdU antibody was from Rockland Inc., Gilbertsville, PA. Anti-rabbit and anti-mouse antibodies linked to horseradish peroxidase, Alexa-488 and Alexa-594 were from KPL Inc., Gaithersburg, MD or Molecular Probes, Eugene, OR, respectively. InSolution TM ATM kinase inhibitor (KU-55933) was from EMD Chemicals, San Diego, CA. Cytotoxicity assay. HMVEC-d cells were tested for their viability in the presence of varying concentrations (1μM, 5μM and 10μM) of ATM Kinase inhibitor (KU-55933) or upon si-control and si-h2ax transfection using a lactate dehydrogenase cytotoxicity assay (CytoTox 96 Non-Radioactive Cytotoxicity Assay) kit as per manufacturer s instructions (Promega, Madison, WI). RNA interference using sirna transfection. Transfection of primary HMVEC-d cells with sirna was performed using the Neon transfection system (Invitrogen) according to the manufacturer s instructions. Briefly, subconfluent cells were harvested and washed once with 1xPBS and resuspended at a density of

10 cells/ml in resuspension buffer R (provided by the company). 10μl of this cell suspension was mixed with 100 pmol of sirna and then microporated at room temperature using a single pulse of 1350V for 30 ms. After microporation, cells were distributed into complete medium and placed at 37 C in a humidified 5% CO 2 atmosphere. 48 h post-transfection, cells were analyzed for knockdown efficiency by western blotting. All sirna oligonucleotides (sigenome SMARTpool) for H2AX, CHK1, CHK2, c-cbl and non-targeting sirna pool no. 2 were purchased from Thermo Scientific (Catalog no. M , M , M , M and D , respectively). Quantitative real-time reverse transcription-pcr. Gene expression of H2AX was examined by real-time RT-PCR using a SYBR green detection system. The expression levels of H2AX were normalized to Tubulin gene expression. The final mrna levels of the genes studied were calculated using the comparative cycle threshold (Ct) method. The following forward and reverse primers were used for H2AX RT-PCR analysis: 5 -ACTCAACTCGGCAATCCAAG-3 and 5 - GGGTTAGCTGCAGAATTCCA-3, respectively. Measurement of KSHV nuclear entry by real-time DNA polymerase chain reaction (PCR). HMVEC-d cells were infected with KSHV (30 DNA copies/cell) at 37 C for 1h. For measuring KSHV nuclear entry kinetics, pure nuclear isolation from the HMVEC-d cells was performed using a Nuclei EZ Prep nuclei isolation kit (Sigma) following the manufacturer's instructions. Briefly, HMVEC-d cells were infected with KSHV for 2 h, washed, treated with trypsin-edta to remove noninternalized virus, and incubated for varying times of infection. Cells were then lysed on ice for 5 min with a mild lysis buffer (Sigma), and nuclei were concentrated by centrifugation at 500 g for 5 min. Cytoskeletal components loosely bound to the 10

11 nuclei were removed from the nuclear pellet by a repeat of the lysis and centrifugation procedures as described previously (31). Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR (29). The KSHV ORF73 gene cloned in the pgem-t vector (Promega) was used for the external standard. The cycle threshold (Ct) values were used to generate the standard curve and to calculate the relative copy numbers of viral DNA in the samples. Measurement of KSHV gene expression by real-time reverse transcription PCR (RT-PCR). Total RNA was prepared from infected or uninfected cells using an RNeasy kit (Qiagen, Germantown, MD) as described previously (29). To quantitate viral gene expression, total RNA was subjected to ORF73 expression by real-time RT-PCR using gene specific primers and Taqman probes. The relative copy numbers of the transcripts were calculated from the standard curve using the Ct values of different dilutions of in vitro-transcribed transcripts. These values were normalized to GAPDH control reactions. To obtain p values between DMSO and ATM kinase inhibitor treated or si-control and sirna treated cells, an unpaired student s t-test was used. Western blot analysis. The cells were lysed in RIPA lysis buffer containing a protease inhibitor cocktail. Equal amounts of protein samples were resolved by 10-20% SDS-PAGE, and subjected to Western blotting. The immunoreactive bands were developed by enhanced chemiluminescence reaction (NEN Life Sciences Products, Boston, MA) and quantified by following standard protocols (32). The bands were scanned and quantitated using the FluorChemFC2 and Alpha-Imager (Alpha Innotech Corporation, San Leonardo, CA). Immunoprecipitation. Two hundred (200) μg of clarified and pre-cleared cell lysates were incubated overnight with immunoprecipitating antibody at 4 C. The resulting 11

12 immune complexes captured by Protein A-Sepharose were analyzed by Western blots using specific detection antibodies. Immunofluorescence microscopy (IFA). HMVEC-d cells seeded on 8 well chamber slides (Nalgene Nunc International, Naperville, IL) were used. Cells infected with live-kshv or UV-KSHV (30 DNA copies/cell) were fixed for 10 min with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 5 min, washed and blocked with Image-iT FX signal enhancer (Invitrogen) for 20 min. Uninfected and KSHV infected cells were incubated with primary anti-h2ax antibodies overnight at 4 C or for 2 h at 37 C for anti-lana-1 antibodies, followed by fluorescent dyeconjugated secondary antibodies for 1 h at 37 C. For detecting BrdU labeled virus, post fixation step, cells were treated with a 4 N HCl solution (diluted in water) for 10 min at RT to expose the BrdU residues for staining. The cells were then washed several times with PBS, permeabilized with 0.2% Triton X-100 for 5 min and blocked with Image-iT FX signal enhancer (Invitrogen) for 20 min. Cells were then stained with a rabbit anti-brdu antibody, followed by detection with fluorescent dyeconjugated secondary antibody. The Nikon Eclipse 80i fluorescence microscope was used for imaging and analysis was performed using Metamorph imaging software. All experiments were performed at least three independent times. 12

13 RESULTS De novo KSHV infection of primary HMVEC-d cells induces DNA Damage Response (DDR) signaling. KSHV infects in vitro a number of adherent target cells which include endothelial HMVEC-d, HUVEC (umbilical vein endothelial), fibroblast (HFF), and epithelial (293) cells. Entry and infection of KSHV in the in vitro target cells is a very rapid process and viral DNA is detected in the infected cell nuclei as early as 30 min (0.5h) post-infection (p.i.) (3). The virion associated KSHV ds-dna genome is linear, epigenetically naïve, with nicks and breaks, and devoid of DNA methylation and histones (4, 5). Soon after DNA entry into the nucleus, the genome circularizes and associates with the histones and other nucleosome proteins leading to epigenetic control of the HHV-8 genome (33). To determine whether nuclear entry of viral DNA during de novo KSHV infection in primary HMVEC-d cells induces any modulation in the host cell s DDR, we assessed the phosphorylation state of DDR-associated proteins by Western blot analysis. To assess the efficiency of early events of KSHV, we examined the phosphorylation of ERK1/2 which is one of the early signaling events that ensues upon de novo KSHV infection (27). We observed about a 4-fold increase in ERK1/2 phosphorylation as early as 30 min p.i. of HMVEC-d cells which was sustained at 3h p.i. as well (Fig 1A, panel 1, lane 2 and 3). Compared to uninfected cells, a 3.6 and 5.8-fold increase in ATM phosphorylation at serine 1981 was observed at 30 min (0.5h) and 3h p.i., respectively (Fig. 1A, panel 3, lanes 2 and 3). There was no significant change in the total protein levels of ATM (Fig. 1A, panel 4, lanes 1-3). Since, phosphorylation of ATM at serine 1981 induces its kinase activity, we investigated further the phosphorylation of downstream effector proteins involved in 13

14 DDR signaling at different time s post-kshv infection. Compared to uninfected cells, phosphorylation of H2AX at serine 139 ( H2AX) increased to 2.6-fold by 30 min (0.5h) p.i., and continued to be induced throughout the observed 12h p.i. (Fig. 1B, panel 1, lanes 2-6). Interestingly, compared to uninfected cells, we observed an increase in the total H2AX protein levels by about 2.2-fold at 30 min (0.5h) p.i. which was sustained during the 12h p.i. (Fig. 1B, panel 2). In addition, we also observed increased phosphorylation of CHK1 at serine 296 and CHK2 at threonine 68 which reached a maximum of 4.6-fold at 6h p.i. for CHK1 and 7.3-fold at 3h p.i. for CHK2 (Fig. 1B, panels 3 and 5). Similarly, increased phosphorylation of BRCA1 at serine 1524 was detected with about 1.9-fold at 30 min (0.5h) p.i. which declined subsequently (Fig. 1B, panel 7). However, no increase in total protein levels of CHK1, CHK2 and BRCA1 was observed (Fig. 1B, panels 4, 6 and 8), and unlike H2AX, phosphorylation of CHK1, CHK2 and BRCA1 was not protracted and did not significantly increase at later time points of infection. Real time RT-PCR analysis of H2AX mrna expression did not demonstrate any increase at different times post-kshv infection (Fig. 1C), which demonstrated that the observed increase in the total H2AX protein levels was not due to an increase in H2AX gene expression. The typical polyubiquitin chains include either Lys(K48)-linked chains that have been studied in the context of protein degradation (34) or Lys(K63)-linked chains which have been implicated in a variety of non-proteolytic functions including mediators of novel signaling and stability of proteins (35). To determine whether the observed H2AX total protein level increase was due to post-translational modifications affecting its stability, we immunoprecipitated the uninfected and infected cell lysates with anti-h2ax antibodies and reacted with monoclonal 14

15 antibodies specific for K48-linked or K63-linked polyubiquitin chains to determine the status of H2AX polyubiquitination. As shown in figure 1D, lanes 1 and 2 of the top panel (lower exposure) and second panel (higher exposure), compared to uninfected cells, the K48-linkage of H2AX decreased by 3h p.i.. In contrast, the K63- linkage increased in the infected cells (Fig. 1D, panel 3, lanes 1 and 2). Blots were stripped and re-probed with anti-h2ax antibodies, which corroborated with the previously observed results showing an increase in total protein levels of H2AX in the infected cells (Fig. 1D, panel 4, lane 2). These results demonstrated that KSHV infection induces a decrease in K48-linked polyubiquitination and an increase in the K63-linked polyubiquitination of H2AX early during infection. Taken together, these studies demonstrated that de novo KSHV infection induces rapid DDR signaling resulting in the phosphorylation and ubiquitination of H2AX, a functionally active form that is shown to bind to DNA breaks during DDR signaling (36). Live-KSHV and UV inactivated-kshv induce ATM and H2AX phosphorylation early during de novo infection of HMVEC-d cells We next investigated whether infection induced DDR signaling was due to KSHV gene expression. We have shown previously that similar to live-kshv (functional), UV-inactivated KSHV also binds to the cell surface, induces the host cell s pre-existing FAK, Src, PI-3K, NF- B and ERK1/2 signal pathways, enters the target cells efficiently but fails to establish successful infection as viral genes are not expressed (27). To determine whether infection with UV-KSHV results in the delivery of viral genome into the nucleus, HMVEC-d cells were infected for different time points with 30 DNA copies/cell of live KSHV and UV-KSHV. Nuclei from these cells were isolated and the kinetics of nuclei associated genomes determined by 15

16 real-time DNA-PCR. Both live-kshv and UV-KSHV genomes were detected in the infected cell nuclei as early as 30 min (0.5h) p.i. (Figs. 2A and 2B), and roughly 46% fewer UV-KSHV DNA copies compared to live KSHV DNA copies were detected. In addition, nuclei associated UV-KSHV DNA copies also decreased by about 50% at 12h p.i. compared to the level at 30 min (0.5h) p.i. (Fig. 2B). In contrast, similar levels of live-kshv genomes were observed at 6 and 12h p.i. (Fig.2.A). Efficiency of infection was confirmed by analyzing for KSHV-induced phosphorylation of ERK1/2, which was similar in live-kshv and UV-KSHV infected HMVEC-d cells (Fig 2C, panel 1, lane 2 and 3). Infection by both live-kshv and UV-KSHV induced similar levels of increase in ATM phosphorylation at 3h p.i. (Fig. 2C, panel 3, lanes 2 and 3, respectively), and H2AX phosphorylation between 30 min (0.5h) to 12h p.i. (Fig. 2D, panel 1, lanes 2-6 and lanes 8-12, respectively), as well as an increase in total H2AX levels (Fig. 2D). Phosphorylation of ATM and H2AX by UV-KSHV as early as 30 min (0.5h) p.i. suggested that the induction of DDR signaling events were probably initiated by virion, viral components or viral DNA during virus entry by endocytosis and/or nuclear delivery, and that KSHV gene expression is dispensable for DDR activation at the early times of infection. H2AX colocalizes with the KSHV genome early during de novo infection of HMVEC-d cells The hallmark of DDR signal initiation is the phosphorylation of H2AX at serine 139 ( H2AX) which is the functionally active form binding to DNA breaks and considered a biomarker of DNA damage (11). Since we observed an increase in H2AX phosphorylation during de novo infection with UV-KSHV, we next investigated whether the KSHV genome is recognized and gets associated with 16

17 H2AX. To visualize the immediate-early events of viral infection, cells were infected for varying time intervals with purified KSHV virions with bromodeoxyuridine- (BrdU) labeled genome. These cells were examined by immunofluorescence assay (IFA) for the association of viral genome with H2AX that forms distinct nuclear foci upon recognition of DNA breaks (37). By 15 min p.i., viral particles detected by rabbit anti-brdu antibodies can be seen in the cytoplasm and near the nuclear periphery but with no significant signal in the nuclei (Fig. 3, panel 2, red arrows). The specificities of these reactions were demonstrated by the absence of BrdU staining in the uninfected cells (Fig. 3A, panel 1). We also did not observe any H2AX spots in either the uninfected or 15 min infected cell s nuclei. This suggested that virion entry and signals associated with entry including ROS (3, 38) are not involved in the activation of DDR responses in the infected cells. By 30 min (0.5h) p.i., viral DNA was observed in the perinuclear area and significant accumulation of viral DNA was observed by 3h p.i. in the nuclei of infected cells (Fig. 3A, panels 3 and 4), which was consistent with the results in Figs. 2A and 2B. Moreover, formation of distinct H2AX nuclear foci was also observed in the infected cells as early as 30 min (0.5h) p.i., which colocalized with the viral genome (Fig. 3A, panels 3 and 4, yellow arrows). Notably, formation of H2AX nuclear foci was not observed in cells that were not infected with KSHV (Fig. 3A, panels 3 and 4, white arrows), which also demonstrated the specificity of the observed H2AX interactions with viral genome. Collectively, these observations demonstrated that during de novo infection of primary HMVEC-d cells, KSHV DNA entering the nuclei initiated the DDR resulting in the phosphorylation of H2AX, colocalization with the KSHV genome and the formation of distinct nuclear H2AX foci. 17

18 Live-KSHV and UV-KSHV induces formation of H2AX nuclear foci early during de novo infection that ceases in UV-KSHV infected cells at later times Next, we determined whether the KSHV-induced H2AX nuclear foci formation is dependent on viral gene expression. As shown in Fig. 4A top six panels (yellow arrows) and Fig. 4B, compared to uninfected cells, in cells infected with both live and UV-KSHV, similar numbers of H2AX nuclear foci were observed from 0.5h to 12h p.i.. However, while a similar number of the H2AX nuclear foci persisted at 24h and 48h p.i. in live-kshv infected cells (Fig. 4A, last two panels, yellow arrows, and Fig. 4B), we observed significantly (p=003) lower numbers of these foci in UV-KSHV infected cells (Fig. 4A, last two panels, white arrows, and Fig. 4B) at 24h to 48h p.i.. Similarly, compared to live-kshv infected cells (Fig. 4C, panel 1, lanes 2 and 5), H2AX phosphorylation was much lower in UV-KSHV infected cells at 24 and 48h p.i. (Fig. 4C, panel 1, lanes 3 and 6), while the total H2AX protein level did not increase. The observed biphasic DDR signaling during de novo KSHV infection suggested that the DDR induction at earlier times of infection is not dependent on KSHV gene expression and is probably induced by the recognition of KSHV DNA entering the nucleus, whereas upon latency establishment, expression of latent viral genes and/or amplification of viral DNA is necessary for the continuous induction of DDR signaling. Inhibition of ATM kinase activity by KU decreases KSHV-induced H2AX phosphorylation and latent ORF73 gene expression To determine the role of ATM kinase activity in KSHV infection-induced H2AX phosphorylation, we utilized KU that specifically inhibits the ATM kinase activity at a very low μm concentration (39). An LDH-based cytotoxicity 18

19 assay demonstrated that incubation of HMVEC-d cells for 2h with 1, 5 and 10 μm concentrations of KU was not cytotoxic (Fig. 5A). Pretreatment of cells with 5μM of KU for 2h reduced the 3h KSHV infection-induced H2AX phosphorylation at 3h from 2.6-fold in DMSO (control) treated to 1.3-fold (Fig. 5B, panel 1, lanes 2 and 4). Interestingly, we did not observe any decrease in the KSHVinduced increase in total H2AX protein levels by KU (Fig. 5B, panel 2, lanes 2 and 4). Similarly, in comparison to ATM (+) G-361 melanoma cells, when ATM (-) HT-144 melanoma cells were infected with KSHV, we observed a reduction in H2AX phosphorylation from 5.9 to 1.2 fold, respectively (Fig 5C, panel 3, lane 3 and 4), but no apparent decrease in total protein levels of H2AX was observed (Fig 5C, panel 4, lane 3 and 4). These results suggested that KSHV-induced phosphorylation of H2AX is ATM dependent, and in contrast, the increase in total protein levels of H2AX is independent of ATM kinase activity. Efficiency of KSHV infection was confirmed by analyzing for KSHV-induced phosphorylation of ERK1/2, which was similar in KSHV infected ATM positive and negative cells (Fig 5C, panel 1, lane 3 and 4). To determine whether ATM-mediated DDR plays roles in KSHV biology, the effect of ATM kinase inhibition on KSHV latent ORF 73 (LANA-1) gene expression was examined. Pretreatment of cells with 5μM of KU for 2h resulted in >50% reduction in ORF73 expression at 48h p.i., as determined by real-time RT-PCR (Fig. 5D). Similarly, when ATM (-) HT-144 melanoma cells were infected, we observed >50% reduction in ORF73 gene expression compared to ATM (+) G-361 melanoma cells at 48h p.i. (Fig. 5E). This reduction was not due to a defect in KSHV entry and nuclear delivery of virion DNA since similar viral DNA copy numbers were detected in the infected nuclei of ATM (-) and ATM (+) cells as determined by real-time DNA-PCR analysis (Fig. 5F). In addition, when compared to ATM (+) cells, we also 19

20 observed >70% reduction (p= ) in the infected cell nuclei associated KSHV DNA copy numbers in ATM (-) cells at 48 h p.i. (Fig. 5G). Moreover, in congruence to our observed results, inhibition of ATM kinase activity resulted in a significant (p= ) decrease in KSHV-induced LANA-1 puncta, a mark of latency establishment detected by IFA analysis (Figs. 6A and 6B). Taken together, our results suggested that ATM induction during de novo KSHV infection of primary HMVEC-d cells plays an important role in the establishment of KSHV latency. Knockdown of H2AX decreases latent ORF73 expression, viral DNA copy number and establishment of KSHV latent infection in HMVEC-d cells KSHV infection induced formation of H2AX nuclear foci and their colocalization with KSHV genome prompted us to investigate its role in infection. When sirnas were microporated into the primary HMVEC-d cells, si-h2ax knocked down >90 % H2AX at the protein level as compared to si-control transfected cells (Fig. 7A, panel 1, lanes 2 and 3). Knocking down of c-cbl, a molecule that is critical for KSHV entry into HMVEC-d cells (40, 41), served as a control (Fig. 7A, panel 2, lane 4). Specificities of the sirnas were shown by the absence of si-h2ax off-target effects on c-cbl and actin, and no effect by si-ccbl over H2AX (Fig. 7A, panel 2). Compared to si-control transfected cells, knockdown of H2AX resulted in >70% reduction in ORF73 gene expression at 48h p.i. (Fig. 7B). As expected, expression of ORF73 in si-c-cbl transfected cells was completely abolished (Fig. 7B), as no viral entry occurred in these cells (40, 41). Successful knockdown of H2AX (Fig. 7E) also resulted in a significant (p=0001) decrease in LANA-1 puncta (Figs. 7C and 7D). 20

21 To rule out the possibility that the observed effects of H2AX knockdown on ORF 73 gene expression were not due to its effect on the viability of cells, a cytotoxicity assay upon si-transfection was performed. We did not observe any appreciable difference in the cellular toxicity among non-transfected, mock, si-control or si-h2ax-transfected cells (Fig. 8A and 8B). Next, we determined the kinetics of ORF 73 gene expression during H2AX knockdown. Compared to control transfected cells, ORF73 gene expression was significantly reduced by H2AX knockdown at 24h p.i. (Fig. 8C). Compared to si-control cells, we also observed >80% reduction (p=0.009) in the infected cell nuclei associated KSHV DNA copy numbers in si- H2AX HMVEC-d cells (Fig. 8D). Collectively, our results demonstrated that in addition to KSHV-induced ATM, H2AX ( H2AX), a downstream signaling molecule of ATM, plays an important role in the establishment of KSHV latency during de novo infection of primary HMVEC-d cells. Knockdown of CHK1 and CHK2 does not affect ORF73 expression in HMVECd cells To investigate the potential role of DDR signaling effector proteins, such as CHK1 and CHK2, that were transiently phosphorylated during KSHV infection (Fig. 1B, panel 3 and 5), we performed knockdown assays and determined their effect on KSHV infection. Robust knockdown efficiency of si-chk1, si-chk2 or cotransfected si-chk1 and si-chk2 was confirmed by western blot analysis (Fig. 9A, panel 1 and 2, lanes 3-5). In contrast to knockdown of H2AX, knockdown of neither CHK1 nor CHK2 or the combination of both, did not show any effect on ORF73 gene expression (Fig. 9B). We also explored the potential consequences of KSHV-induced CHK1 and CHK2 kinase activation by examining the phosphorylation of Cdc25c, a 21

22 downstream substrate that acts as a phosphatase and has a role in cell cycle control regulation (42, 43). By western blot analysis, we did not observe any alteration in the phosphorylation of Cdc25c at serine 216 (Fig. 9C, panel 1, lanes 1-6). These results suggested that KSHV infection induces a selective arm of the DDR, such as ATM and H2AX, for its survival advantage. Downloaded from on October 1, 2018 by guest 22

23 DISCUSSION All herpesviruses, as obligate intracellular parasites maintaining their latent life cycle in the nucleus of infected cells, rely on and often manipulate host cell replication and repair factors for their own benefit. In the early period of KSHV infection, upon nuclear entry of virion-associated epigenetically naïve, linear dsdna with nicks and breaks and devoid of DNA associated proteins such as histones, a dynamic event ensues involving the association of many of the host s cellular factors with the viral genome followed by the expression of viral genes. These events are highly regulated and require the coordinated involvement of cellular epigenetic machinery (44). Upon establishment of latency, KSHV persists in the host cell nuclei without lytic replication and maintains an episomal form of viral genome with minimal gene expression. Delineating the changes in the cellular environment during the early stages of de novo KSHV infection can provide important clues to the mechanisms regulating these dynamic processes during primary infection as well as latency. Our findings here demonstrate that the KSHV genome upon its entry into the nuclei of an infected cell triggers a rapid DDR by the activation of cellular ATM kinase activity, sustained increase in phosphorylation and total protein levels of H2AX (Fig. 10), and transient increase in the phosphorylation of CHK1 and CHK2. Increase in total protein levels of H2AX at the early stage of infection were not due to an increase in its gene expression. Instead, the observed decrease in the Lys 48-linked polyubiquitination of H2AX involved in protein degradation (34) with concomitant increase in the non-proteolytic Lys 63-linked polyubiquitination demonstrated that KSHV infection induces H2AX stabilization leading into the rapid increase in the total protein levels of H2AX. Experiments with antibodies specific to distinct 23

24 ubiquitin chains have established that DNA-damage-flanking chromatin is enriched in Lys 63-linked ubiquitin polymers (45). ATM-mediated phosphorylation of Mediator of DNA-Damage Checkpoint 1 (Mdc1) triggers recruitment of RNF8, a RINGdomain ubiquitin ligase to initiate a ubiquitination cascade of proteins involved in the repair of DNA damage (45). H2AX is polyubiquitinated by RNF8 and in association with another ubiquitin ligase RNF168 (46-48). Inhibition of ATM kinase activity with a selective inhibitor KU55933 significantly inhibited the KSHV-induced phosphorylation of H2AX, however, it did not affect the increase in total protein levels of H2AX (Fig. 5B). Similarly, de novo infection of ATM (+) and ATM (-) cells further demonstrated that KSHV-induced H2AX phosphorylation is ATM dependent, however, the KSHV-induced increase in total H2AX levels is ATM independent (Fig 5D). These results suggest an involvement of another signaling event that is independent of ATM kinase activity in H2AX stability, which needs to be examined further. We believe that to establish a potential link between the DDR associated ATM and H2AX with other KSHV-induced early signaling events, need additional extensive studies such as experiments employing small molecule inhibitors and knockdown approaches which are beyond the scope of the present manuscript. Studies by Tarakanova et al., demonstrating that MHV68 ORF36 and its homologue BGLF4 of Epstein-Barr virus (EBV) phosphorylate H2AX during infection (24) suggest that -herpesviruses can actively initiate the host DDR by their kinases. Moreover, interplay of viral proteins with the regulatory ubiquitination arm of the DDR machinery has also been previously reported for HSV-1, wherein, viral ubiquitin ligase, ICP0 has been shown to target both RNF8 and RNF168 for proteasome-mediated degradation, thereby preventing the deposition of repressive ubiquitin marks and counteracting the recruitment of repair proteins 53BP1 and 24

25 BRCA1, but not H2AX or Mdc1 to the incoming viral genomes (49). Further studies are essential to determine whether an analogous mechanism exists in KSHV at the early stages of infection that selectively exploits the important ubiquitination arm of the DDR machinery. The rapid activation of KSHV-induced DDR culminated in the formation of H2AX foci and was independent of viral gene expression at the early stages of infection. Furthermore, upon entry of the KSHV genome into the nucleus by 30 min (0.5h) p.i., formation and colocalization of H2AX foci with the viral genome was also observed. Similar immunofluorescent colocalization studies have shown that H2AX localized in foci juxtaposed to parental HCMV DNA (Towne strain) at early times of infection in human fibroblasts (20). Moreover, H2AX accumulates at the intranuclear sites of HCMV replication during the late stages of infection (20), forming distinct foci that appear to surround the viral inclusions. In contrast, H2AX did not colocalize with late HSV-1 replication compartments (18), although it accumulated at sites associated with the incoming HSV-1 genome (49). Posttranslational modifications of H2AX, notably, phosphorylation and ubiquitination have been believed to be providing the docking sites and function as a platform in recruitment and loading of DNA-damage repair proteins to the chromatin. It is reasonable to speculate that association of H2AX with the KSHV genome may also provide a similar environment in recruiting chromatin modifier proteins to the newly entered viral DNA leading into the reported extensive chromatin modifications (4, 44) that are essential for successful maintenance of KSHV DNA as an episome and consequently in the establishment of latency. KSHV viral latent proteins have been shown to induce H2AX phosphorylation (21, 22), and phosphorylation of H2AX has been observed in KSHV positive KS 25

26 lesions (21). Corroborating those reports, our present results demonstrate that KSHVinduced phosphorylation of H2AX observed during the late stages of de novo infection in primary endothelial cells is dependent on viral gene expression as UVinactivated KSHV could not induce such response at late time points of infection. We also observed an increase in H2AX phosphorylation in the KSHV latently infected primary effusion lymphoma (BCBL-1) and long term culture of telomere immortalized vascular endothelial (TIVE-LTC) cells, compared to uninfected B-cell lymphoma BJAB and TIVE cells, respectively (data not shown). Taken together, these results suggest that latent viral gene expression and/or amplification of viral DNA is necessary for the sustained induction of KSHV-induced DDR signaling upon de novo infection. Recently, it has been shown that de novo KSHV infection of human peripheral blood mononuclear cells (PBMCs) lead to an increase in H2AX phosphorylation at 2 and 6 day p.i.. Interestingly, phosphorylated H2AX localized to KSHV Terminal Repeats (TRs) contribute to binding of LANA-1 to the TRs and consequently in the LANA-mediated KSHV episome persistence in stably infected HEK293 cells (22). Our results demonstrated that inhibition of ATM kinase activity that abrogated H2AX phosphorylation or depletion of H2AX reduced the expression of viral latent proteins and consequently establishment of viral latency. This suggests that components of DDR signaling are critical during the initial stages of KSHV infection (Fig. 10). Interestingly, we also observed a decrease in the viral DNA copy number in H2AX depleted cells which corroborated the results of a previous study (22). Together with the demonstrated role of H2AX in tethering the KSHV episome to LANA-1 and colocalization of H2AX with LANA-1 in PEL-derived B-cell lymphoma cells (BC-3 and BCBL-1) (22), our present studies of de novo infection 26

27 suggest that H2AX contributes to the persistence of the KSHV genome, and its induction is essential very early during the de novo infection of primary endothelial cells. We propose that the absence of tethering of the viral genome to the host chromosome mediated by H2AX/LANA-1 during primary infection may lead into several consequences such as the observed reduced latent gene expression, and/or reduction in genome copy numbers due to its susceptibility to yet to be identified host factors targeting non-chromosomal DNA. Further studies are essential to explore these potential possibilities. Downregulation of downstream CHK1 and CHK2 proteins did not affect viral latent gene expression, establishment of viral latency, or the CHK1- and CHK2- induced signaling cascade of cell cycle progression (Fig. 9C). Collectively, our findings suggest that KSHV does not induce a full-scale DDR which may lead into the activation of signaling that might prove detrimental to the establishment of KSHV infection, and instead, induces selective DDR pathways for its own advantage. It can be envisioned that selective perturbations of DDR signaling by KSHV, a tumorassociated virus, may consequently lead to cellular tumorigenesis. Targeting ATM and H2AX could serve as an attractive strategy to block the establishment of KSHV latent infection and the associated malignancies. ACKNOWLEDGEMENTS This study was supported in part by Public Health Service Grants CA , CA and CA to BC and RFUMS-H.M. Bligh Cancer Research Fund to BC. We thank Keith Philibert for critically reading this manuscript. 27

28 FIGURE LEGENDS Figure 1. Induction of DNA Damage Response (DDR) signaling and effect on the mrna expression of H2AX during de novo KSHV infection of HMVEC-d cells. (A) Primary endothelial (HMVEC-d) cells infected with KSHV (30 DNA copies/cell) were analyzed for ERK1/2 and ATM phosphorylation and their total protein levels by immunoblot analysis. Fold changes were calculated considering levels of uninfected cells as 1. (B) HMVEC-d cells infected with KSHV (30 DNA copies/cell) for varying time points of early stages of infection were analyzed for phosphorylation of H2AX, CHK1, CHK2 and BRCA1 along with their total protein levels by immunoblot analysis. -Tubulin and -actin and were used as loading controls. Fold changes were calculated considering levels of uninfected cells as 1. (C) Primary HMVEC-d cells infected with KSHV (30 DNA copies/cell) for varying time points were analyzed for H2AX gene expression by real-time RT-PCR. Each bar represents the fold increase in gene expression ± SD of three independent experiments. Fold changes were calculated considering levels of uninfected cells as 1 after normalizing with expression of the -tubulin gene. (D) Primary HMVEC-d cells infected with KSHV for 3h were analyzed for KSHV-induced poly-ubiquitination of H2AX. Immunoprecipitation of 200 g of the whole-cell lysate (WCL) was carried out with rabbit anti-h2ax antibodies and subjected to Western blot analysis for either Lys-48 or Lys-63 specific poly-ubiquitination. Blots were stripped and reprobed with H2AX. Black Arrows indicate poly-ubiquitination (K48-1 st and 2 nd panel) and (K63-3 rd panel) of H2AX. Figure 2. Induction of ATM and H2AX phosphorylation during de novo infection of HMVEC-d cells with live and UV-inactivated KSHV. (A and B) HMVEC-d 28

29 cells infected with KSHV or UV-KSHV (30 DNA copies/cell) were analyzed for kinetics of nuclear delivery of KSHV genome by real-time DNA-PCR of isolated nuclei from uninfected or infected cells. ORF 73 standards and non-template controls were run in parallel. The viral DNA copy numbers were calculated from a standard graph generated by real-time DNA-PCRs of known concentrations of a cloned ORF73 gene. Each reaction was done in triplicate, and each bar represents the mean ± SD for three experiments. (C) HMVEC-d cells infected with KSHV or UV-KSHV for 3h were analyzed for phosphorylation of ERK1/2 and ATM along with their total protein levels by immunoblot analysis. Fold changes were calculated considering levels of uninfected cells as 1. (D) HMVEC-d cells infected with KSHV or UV-KSHV for the indicated time points were analyzed for phosphorylation of H2AX and total protein levels by immunoblot analysis. -actin was used as loading control. Fold changes were calculated considering levels of uninfected cells as 1. Figure 3. Immunofluorescence analysis of H2AX colocalization with BrdU labeled KSHV genome early during de novo infection of HMVEC-d cells. (A) HMVEC-d cells were infected with BrdU labeled KSHV (30 DNA copies/cell) for 2h, uninternalized virus removed by washing, and infected and uninfected cells further incubated at 37 C for the indicated time points. The cells were washed, fixed, permeabilized and blocked with Image-iT FX signal enhancer. Cells were stained with anti-brdu and anti- H2AX antibodies and visualized by incubation with Alexa- 488 (green) and Alexa-594 (red) secondary antibodies, respectively. The image was merged with DAPI stained nuclei. The boxed areas were enlarged and presented in the right most panels. Yellow arrows represent the H2AX foci colocalizing with KSHV genome in the infected cells. Red arrows represent the KSHV genome not 29

30 internalized in the nucleus while white arrows represent uninfected cells. Image results are depicted from a representative field taken from three independent experiments. Magnification: 60X. (B) The percentage colocalization of H2AX-foci per infected cell is represented in the graphical plot. A minimum of 3 fields having at least 15 cells were chosen and the error bars show mean ± SD. Figure 4. Immunofluorescence analysis of H2AX levels early during de novo infection of HMVEC-d cells by live-kshv and UV-KSHV. (A) HMVEC-d cells were infected with KSHV (30 DNA copies/cell) for 2h, uninternalized virus removed by washing, and infected and uninfected cells further incubated at 37 C for the indicated time points. The cells were washed, fixed, permeabilized and blocked with Image-iT FX signal enhancer. Cells were stained with anti- H2AX antibodies and visualized by incubation with Alexa-594 (red) secondary antibodies. The image was merged with DAPI stained nuclei. The boxed areas were enlarged and presented in the right most panels. Yellow arrows represent the H2AX foci in the KSHV infected cells and white arrows represent the decrease in H2AX foci formation in the UV- KSHV infected cells. Image results are depicted from a representative field from three independent experiments. Magnification: 60X. (B) The number of H2AX-foci per cell is represented in the graphical plot. A minimum of 3 fields having at least 15 cells were chosen and the error bars show means ± SD. (C) HMVEC-d cells infected with live-kshv or UV-KSHV (30 DNA copies/cell) for the indicated times were analyzed for phosphorylation of H2AX and total protein levels by immunoblot analysis. -actin was used as loading control. Fold changes were calculated considering levels of uninfected cells as 1. 30

31 Figure 5. Effect of ATM kinase activity inhibition by KU on KSHVinduced phosphorylation of H2AX and latent ORF73 gene expression during de novo infection of HMVEC-d cells. (A) Supernatants of HMVEC-d cells treated with DMSO or 1μM, 5μM and 10μM concentrations of ATM kinase inhibitor for 2h were analyzed for their effect on cellular viability by LDH-based toxicity assay. (B) HMVEC-d cells pretreated with DMSO or ATM kinase inhibitor for 2h were infected with KSHV (30 DNA copies/cell). 3h post-infection, cellular lysates were analyzed for phosphorylation of H2AX and total protein levels by immunoblot analysis. Fold changes were calculated considering levels of uninfected cells as 1. (C) ATM-positive G-361 (ATCC CRL-1424 TM ) and ATM negative HT-144 (ATCC HTB-63 TM ) melanoma cells infected with KSHV (30 DNA copies/cell) were analyzed for ERK1/2 and H2AX phosphorylation and their total protein levels by immunoblot analysis. Fold changes were calculated considering levels of uninfected cells as 1. (D) DMSO or ATM kinase inhibitor treated HMVEC-d cells for 2h were infected with KSHV. At 48h p.i., cells were harvested for RNA isolation and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Data represented are mean ± SD of three experiments. (E) ATM-positive and ATM negative cells were infected with KSHV for 2 h. At 48 h p.i., cells were harvested for RNA isolation and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Data represented are mean ± SD for three experiments. (F and G) ATM positive and negative cells were infected with KSHV (30 DNA copies/cell) for 3h (F) and 48 h (G). Nuclear delivery of viral genome was determined by real-time DNA-PCR using the isolated nuclei of uninfected or infected cells. Each reaction was done in triplicate, and each bar represents the mean ± SD for three experiments. The viral DNA copy numbers were calculated from a standard 31

32 curve generated by real-time PCRs of known concentrations of a cloned ORF73 gene. Each reaction was done in triplicate, and each bar represents the mean ± SD for three experiments. Figure 6. Effect of inhibition of ATM kinase activity by KU in latency establishment during de novo KSHV infection of HMVEC-d cells. (A) DMSO or ATM kinase inhibitor treated HMVEC-d cells for 2h were infected with KSHV (30 DNA copies/cell), uninternalized virus removed by washing, and infected and uninfected cells further incubated at 37 C for 48 h. The cells were washed, fixed, permeabilized and blocked with Image-iT FX signal enhancer. Cells were stained with anti-lana-1 antibodies and visualized by incubation with Alexa-594 (red) secondary antibodies. The image was merged with DAPI stained nuclei. The boxed areas were enlarged and presented in the right most panels. Image results are depicted from a representative field taken from three independent experiments. Magnification: 60X. (B) The number of characteristic LANA-1 puncta per infected cell is represented in the graphical plot. A minimum of 3 fields having at least 15 cells were chosen and the error bars show mean ± SD. Figure 7. Effect of H2AX knockdown on KSHV ORF73 gene expression and latency establishment during de novo infection of HMVEC-d cells. (A) Knockdown efficiency of si-control, si-h2ax, si-c-cbl transfection in HMVEC-d cells was determined by Western blot. -actin was used as loading control. (B) si- Control, si-h2ax or si-c-cbl transfected HMVEC-d cells were infected with KSHV for 2h. At 48h p.i., cells were harvested for RNA isolation and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Data 32

33 represented are mean ± SD for three experiments. (C) si-control and si-h2ax transfected HMVEC-d cells were infected with KSHV (30 DNA copies/cell) for 2h, uninternalized virus removed by washing, and infected and uninfected cells further incubated at 37 C for the indicated times. The cells were washed, fixed, permeabilized and blocked with Image-iT FX signal enhancer. Cells were stained with anti- LANA-1 antibodies and visualized by incubation with Alexa-594 (red) secondary antibodies. The image was merged with DAPI stained nuclei. The boxed areas were enlarged and presented in the right most panels. Image results are depicted from a representative field taken from three independent experiments. Magnification: 60X. (D) The number of characteristic LANA-1 puncta per infected cell is represented in the graphical plot. A minimum of 3 fields having at least 15 cells were chosen and the error bars show mean ± SD. (E) Knockdown efficiency of si-h2ax transfection in HMVEC-d cells was determined by Western blot. -actin was used as loading control. Figure 8. Effect of H2AX knockdown on kinetics of KSHV ORF73 expression and infected cell nuclei associated viral DNA copy number during de novo infection of HMVEC-d cells. (A) Knockdown efficiency of si-h2ax in HMVEC-d cells was determined by Western blot. -actin was used as loading control. (B) Supernatants of non-transfected, mock, si-control or si-h2ax-transfected HMVEC-d cells were analyzed 48h after transfection for their effect on cellular viability by LDH-based toxicity assay. (C) si-control and si-h2ax transfected HMVEC-d cells were infected with KSHV for 2h. At the indicated times p.i., cells were harvested for RNA isolation and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Data represented are mean ± SD (n = 3). (D) si- Control and si-h2ax transfected HMVEC-d cells were infected with KSHV (30 33

34 DNA copies/cell) for 48h and analyzed for KSHV genome associated with infected cell nuclei by real-time DNA-PCR with ORF73 primers. The viral DNA copy numbers were calculated from a standard graph generated by real-time PCRs of known concentrations of a cloned ORF73 gene. Each reaction was done in triplicate, and each bar represents the mean ± SD for three experiments. Figure 9. Effect of CHK1 and CHK2 knockdown on KSHV ORF73 gene expression during de novo infection of HMVEC-d cells. (A) Knockdown efficiency of si-chk1, si-chk2 or si-chk1 + si-chk2 transfection in HMVEC-d cells was determined by Western blot. -actin was used as loading control. (B) si-control, si- CHK1, si-chk2 or si-chk1 + si-chk2 transfected HMVEC-d cells were infected with KSHV for 2h. At 48 h p.i., cells were harvested for RNA isolation and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Data represented are mean ± SD for three experiments. (C) KSHV (30 DNA copies/cell) infected HMVEC-d cells at different time points were analyzed for phosphorylated Cdc25c and total protein levels by immunoblot analysis. -actin was used as loading control. Figure 10. Schematic representation of KSHV-induced DNA Damage Response (DDR) during de novo infection of endothelial cells and implication in the establishment of infection. During de novo KSHV infection of primary HMVEC-d cells, linear dsdna in the virions delivered into the nuclei as early as 30 min (0.5h) post-infection circularizes. DDR signaling is initiated by the ATM kinase probably by the recognition of nicks and gaps in the viral DNA, which in turn induces the phosphorylation of H2AX ( H2AX) and the formation of distinct multiple H2AX 34

35 foci and association with KSHV genome. Disruption of ATM kinase activity by kinase inhibitor and knockdown of H2AX results in significant reduction in KSHV latent gene expression, infected nuclei associated viral DNA and establishment of KSHV latency. Our results also suggest that latent viral gene expression and/or amplification of viral DNA is necessary for the sustained induction of KSHV-induced DDR signaling upon de novo infection. Together with the demonstrated role of H2AX in tethering the KSHV episome to LANA-1 and colocalization of H2AX with LANA-1 in PEL-derived B- cell lymphoma cells (BC-3 and BCBL-1) (22), our present studies of de novo infection of endothelial cells suggests that ATM and H2AX contribute to the persistence of the KSHV genome, and their induction is essential very early during the de novo infection of primary endothelial cells. We propose that during de novo infection, KSHV does not induce a full-scale DDR which may lead into the activation of signaling that might prove detrimental to the establishment of KSHV infection, and instead, induces a selective arm of the DDR, such as ATM and H2AX, for its survival advantage. 35

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