Mobile lysates were being analyzed by immunoblots with antibodies in opposition to H2AX (S135), ZEBRA, and GAPDH (B: i) or GAPDH and H2AX (B: ii) fold-stimulation values of H2AX normalized to H2AX are indicated. (C) HH514-sixteen cells had been irradiated with Gy, 2Gy, five Gy, 10 Gy, 20 Gy, or treated with AZA. Whole RNA was extracted from cell lysates immediately after 24 h or forty eight h and relative stages of BZLF1 mRNAs were being calculated by qRT-PCR using the regular-curve technique. Specific samples were being assayed in triplicate in two organic replicates.677746-25-7To examine whether ATM kinase action is important for induction of the EBV lytic cycle in HH514-sixteen and Raji we analyzed EBV reactivation in the presence of an ATM kinase inhibitor, KU55933 [46]. To verify that the inhibitor was useful we assayed H2AX induction in the existence or absence of KU55933. Right after 2h of remedy of Raji cells with camptothecin and KU55933, induction of H2AX was inhibited by about 70% in contrast to camptothecin-treated Raji cells (Fig 8A, i). In contrast to Raji cells addressed with TPA, remedy of Raji cells with TPA and KU55933 resulted in a 90% reduction of H2AX activation (Fig 8B, iii), and around seventy five% inhibition of the two EA-D (Fig 8B, i and iv) and ZEBRA (Fig 8B, ii) protein expression. There was 60% inhibition of H2AX induction immediately after remedy of HH514-16 cells with KU55933 and camptothecin for 2h relative to cells dealt with with camptothecin by yourself (Fig 8A, ii). In comparison to HH514-sixteen cells dealt with with AZA, remedy of HH514-sixteen cells with AZA and KU55933 resulted in a ninety% reduction in H2AX activation (Fig 8C, ii), about fifty% inhibition of ZEBRA (Fig 8C, ii), and 70% inhibition of EA-D proteins (Fig 8C, i and iii). These information suggest that ATM kinase action is required for maximal activation of the EBV lytic cycle.ATM kinase activity mediates maximal reactivation of the EBV lytic cycle in Burkitt lymphoma cells. (A) Mobile lysates from Raji (A: i) and HH514-16 cells (A: ii) untreated or taken care of with camptothecin (CAMPTO), in the presence or absence of KU55933, had been analyzed by immunoblots with antibodies versus H2AX (S135) and GAPDH fold induction of H2AX normalized to unphosphorylated H2AX are demonstrated. (B) Mobile lysates of Raji cells untreated, or taken care of with TPA, in the existence or absence of KU55933, ended up analyzed by immunoblots with antibodies against EA-D and GAPDH (B: i), ZEBRA and -Actin (B: ii), and H2AX (S135) and GAPDH (B: iii) The fold stimulation values of EA-D and ZEBRA normalized to GAPDH or H2AX normalized to unphosphorylated H2AX are demonstrated. The average fold-induction of EA-D normalized to GAPDH was determined (n = 3) denotes P<0.01 P = 0.00013 for TPA+KU55933 versus TPA (B: iv). (C) Cell lysates of HH514-16 cells untreated, or treated with AZA, in the presence or absence of KU55933, were analyzed by immunoblots with antibodies against EA-D and GAPDH (C: i) and ZEBRA, H2AX, and -Actin (C: ii) The fold stimulation values of EA-D, ZEBRA, and H2AX are shown. The average fold-induction of EA-D normalized to GAPDH was determined (n = 3). denotes P<0.01 P = 0.00020 for AZA+KU55933 versus AZA (C: iii).Our data in BL cells showed that DNA damage signaling occurs early during the EBV lytic cycle and is not dependent on viral lytic replication. Three EBV proteins expressed during the early lytic cycle, BGLF4, BGLF5, and BALF2 induce markers of DNA damage signaling, including H2AX, in EBV-negative cells [25, 26, 34]. In our experiments, we showed that BGLF4 and BGLF5 proteins were not essential for early lytic phase induction of pATM foci (Figs 2B and 3B). The detection of pATM foci or H2AX in the absence of BGLF4, BGLF5, and BALF2 in lytically-reactivated G4G5 and Raji cell lines led us to conclude that these proteins may not be the only EBV proteins that contribute to induction of markers of DNA damage signaling in the pre-replicative stage of the EBV lytic cycle (Figs 3). Our subsequent studies explored the hypothesis that ZEBRA may activate DNA damage signaling during the EBV lytic cycle. ZEBRA is sufficient and indispensable for EBV lytic reactivation and is expressed during the very early phase of the lytic cycle [47]. Two observations in our previous experiments led us to hypothesize that ZEBRA may play a direct role in activating markers of DNA damage signaling. First, in experiments in 2089 cells, where the EBV lytic cycle was induced by transfection with a ZEBRA expression vector, the percentage of pATM-positive cells containing ZEBRA was greater than the percentage of pATM-positive cells containing Rta (Fig 1C: iii). This finding could be explained if expression of ZEBRA was sufficient to activate pATM in some cells, without activating downstream lytic genes. Secondly, induction of H2AX in HH514-16 cells temporally correlated with expression of ZEBRA (Fig 4E). To determine whether ZEBRA is sufficient for induction of markers of DNA damage signaling, we expressed ZEBRA in 293 cells, the parental cell line for the EBV-bacmid bearing 2089 and G4 G5 cells. pATM foci were detected in a significant proportion (about 30%) of ZEBRA-positive 293 cells (Fig 9A and 9B Fig 10A: ii). We co-transfected 293 cells with CMV and mGFP plasmids or ZEBRA and mGFP plasmids and quantified the proportion of mGFP-positive cells with pATM foci in each sample, in an observer-blinded fashion (Fig 9B). With this blinded approach, there were consistently greater proportions of pATM-positive cells in ZEBRA-transfected cells (Fig 9B). H2AX was also detected by immunoblot in EBV-negative 293 cells expressing ZEBRA (Fig 9C: i and 9D). These data showed that ZEBRA expression is sufficient for induction of pATM and H2AX, biochemical markers of DNA damage signaling. Induction of DNA damage signaling may lead to apoptosis or perturbation of cell cycle progression. Cells expressing ZEBRA were not preferentially stained with 7-aminoactinomycin D (7AAD), which stains apoptotic and dead cells (S4 Fig). These data indicate that cells expressing ZEBRA do not undergo apoptosis or cell death.Some viral transcription factors induce DNA damage in a DNA-binding-dependent manner. We therefore studied ZEBRA mutants with single amino-acid substitutions within the DNA recognition domain that affect binding to a high affinity ZEBRA response element (ZRE), ZIIIB, to determine whether ZEBRA's ability to induce phosphorylated ATM correlated with its DNA binding capacity. We have previously studied DNA binding and lytic cycle activation properties of ZEBRA mutants, including Z(R179A), Z(S186A), Z(S186E), Z(N182E), and Z (R183E) (Table 1 and [48]). Z(R179A) activates expression of Rta but not EA-D or late lytic proteins when expressed in 2089 EBV-positive cells (Table 1). Z(S186A), Z(S186E), Z(N182E), and Z(R183E) mutants are defective in EBV lytic cycle activation. Of these defective mutants, only Z(S186A) binds to the ZIIIB ZEBRA binding DNA site (Table 1 [48]). Although Z (N182E) is defective in binding ZIIIB sites, it binds to AP1 DNA sequences comparably to wild type ZEBRA (S5 Fig). We compared the relative ability of Z(R179A), Z(S186A), Z(S186E), Z(N182E), and Z (R183E) mutants to activate pATM foci relative to wild type ZEBRA. Expression of ZEBRA mutants was similar to that of wild type ZEBRA (S6 Fig). As seen with wild-type ZEBRA (Fig 9A and 9B and Fig 10A:ii), the Z(S186A) (Fig 10A:iii and 10B) and Z(N182E) (Fig 10A:iv and 10B) mutants induced pATM foci in a significant proportion of 293 cells relative to the negative control (Fig 10B). Foci of pATM were also detected in EBV-positive Raji cells expressing the Z(R179A) (Fig 11A:ii) and Z(S186A) (Fig 11A:iii) mutants. However, the proportion of pATM positive cells in response to expression of DNA binding defective mutants Z(S186E) pATM is induced in response to ZEBRA expression in cells lacking EBV. (A) 293 cells were transfected with a plasmid expression vector for ZEBRA. Cells were double-stained for ZEBRA and pATM (S1981). White arrows indicate cells expressing ZEBRA. The blue arrow indicates a cell positive for pATM. Scale bar = 10 m. (B) 293 cells were co-transfected with a plasmid expressing a membrane-targeted EGFPfarnesylated construct (mGFP) and an empty vector (CMV) or an expression vector for ZEBRA and stained for pATM (S1981). The percentage of total cells that were positive for both pATM and mGFP was determined in blinded experiments and the results represented as average percentages (n = 4), denotes P<0.005 P = 0.0048 for ZEBRA versus CMV. (C) 293 cells were transfected with an empty vector (CMV) or a plasmid expression vector for ZEBRA. Cell lysates were analyzed by immunoblots with antibodies against H2AX and ZEBRA and -Actin (C: i), or H2AX and -Actin (C: ii). H2AX fold stimulation (FS) levels are shown n = 3, denotes P<0.005 P = 0.0014 for H2AX FS in CMV versus ZEBRA 293 samples (C: iii)(Fig 10A: v and 10B) and Z(R183E) was not significantly different from negative control cells (Fig 10A: i and 10B). These data provide additional evidence that ATM phosphorylation in response to expression of ZEBRA is not dependent on ZEBRA's ability to activate the EBV lytic cycle, but is related to its ability to bind DNA.We have previously shown that ZEBRA localizes to electron dense regions of the nucleus, which are known sites of heterochromatin [49]. To explore a possible mechanism by which ZEBRA may interact with DNA damage signaling pathways, we examined its intra-nuclear distribution relative to HP1, a heterochromatin associated protein linked to ATM pATM is induced in response to expression of WT ZEBRA, and ZEBRA mutants Z(S186A), and Z(N182E) in 293 cells. (A) 293 cells co-transfected with an empty vector (CMV) and a membrane-targeted EGFP-farnesylated construct (mGFP) (A: i) or singly transfected with expression vectors for ZEBRA (A: ii), Z (S186A) (A: iii), Z(N182E) (A: iv), Z(S186E) (A: v), or Z(R183E) (A: vi) were fixed and double-stained for ZEBRA and pATM (S1981) Arrows indicate transfected cells expressing transfected genes or pATM or transfected genes and pATM. Detector settings were kept constant during confocal image acquisition. (B) 293 cells were fixed and stained for pATM (S1981) after co-transfection with a membrane-targeted EGFPfarnesylated construct (mGFP) and an empty vector (CMV), or expression vectors for ZEBRA, or Z(S186A), or Z(N182E), or Z(S186E), or Z(R183E). The percentage of total cells that were positive for both pATM and mGFP was determined in blinded experiments (n = 4). P<0.01 P = 0.0001 for WTZ vs. CMV, 0.00017 for Z (S186A) vs. CMV, and 0.0013 for Z(N182E) versus CMV phosphorylation [31]. Immuno-electron microscopy with antibodies conjugated to gold particles showed HP1 localization in numerous foci within electron dense regions of 293 cells (Fig 12). ZEBRA co-localized with HP1 in electron dense regions of the nucleus in EBV- negative 293 cells (Fig 12A and 12B). In the presence of ZEBRA, there were significantly fewer HP1 specific gold particles in electron dense regions of the nucleus (Fig 12C). This finding suggests that ZEBRA may displace HP1 from its binding sites on cellular chromatin (see Discussion).Summary of DNA binding activity, EBV lytic gene activation, and DNA damage signaling phenotypes of ZEBRA mutants compared to wild-type ZEBRA. Protein WT ZEBRA Z(R179A) Z(S186A) Z(N182E) Z(S186E) Z(R183E) Binding to ZIIIB sequence + + + BRLF1 gene activation + + BMRF1 gene activation + BFRF3 gene activation + pATM foci capacities for DNA binding and activation of EBV lytic genes by WT and mutant ZEBRA were determined previously [48]. Generation of pATM foci was determined by immunofluorescence assays, in 293 cells, as described in Fig 11. + denotes proficiency, relative to WT, in performing the respective activity. - denotes deficiency, relative to WT, in performing the respective activity.ATM is phosphorylated upon expression of WT ZEBRA, and ZEBRA mutants Z(R179A), and Z (S186A) in Raji cells. (A) Raji cells infected with an empty vector lentivirus (V), or lentiviruses expressing the wild-type ZEBRA gene (A: i), Z(R179A) (A: ii), or Z(S186A) (A: iii) were fixed and double stained for ZEBRA and pATM. Detector settings were kept constant during confocal image acquisition. Scale bar = 10 m. (B) The percentage of total cells that were positive for ZEBRA or ZEBRA and pATM was obtained.ZEBRA localizes to electron dense regions of the nucleus containing HP1. 293 cells were transfected with (A) an empty vector (CMV), or (B) a ZEBRA expression vector, fixed and processed for immunogold labeling with antibodies against ZEBRA and Protein A-conjugated 15 nm gold particles and HP1 and Protein A-conjugated 5 nm gold particles. 2569265The percentage of HP1 clusters with 10 or>10 5nm gold particles was established and represented as signifies (n = 2), 20 nuclei sections had been photographed and scored for the presence of every marker with Mobile Profiler software program. Scale bar = ten m.We show right here that activation of DNA harm response signaling proteins can be elicited in the early period of the EBV lytic cycle and in the absence of EBV DNA replication. Our conclusions lifted two issues: one) which viral variables are dependable for induction of DNA hurt signaling throughout the early stage of the EBV lytic cycle 2) What is the purpose of DNA problems signaling activation affiliated with the early period of the EBV lytic cycle We addressed these queries by investigating the function of early lytic proteins, ZEBRA, BGLF4, BGLF5, and BALF2, in inducing markers of DNA damage signaling and by checking out how the EBV lytic cycle is afflicted by DNA damage signaling.Despite the fact that many EBV proteins, including BGLF4 (Fig 3 and [26]), BGFL5 [34], and BALF2 [twenty five], are ample to induce markers of DNA hurt signaling in EBV-damaging cells, we come across that these proteins are not essential for activation of foci of pATM throughout the EBV lytic cycle (Fig 2B and 2C and Fig six). pATM foci have been detected in G4G5 cells missing the two BGLF4 and BGLF5, on lytic reactivation by expression of ZEBRA (Fig 2B and 2C). In Raji cells, lacking BALF2, H2AX (Fig 4A) and pATM foci (Fig 6A i) ended up induced in cells lytically reactivated by cure with TPA. One particular scenario that may account for the dispensability of BGLF4, BGLF5, BALF2 in activation of pATM or H2AX hinges on the roles these proteins engage in in replication. In the mobile methods we studied, BGLF4, BGLF5, or BALF2 were not needed for inducing DNA hurt signaling events and cells have been predominantly in the pre-replicative phase of EBV. The vast majority of lytically reactivated G4G5 cells did not have replication compartments (Fig 2B and 2C). In Raji cells, thanks to the absence of the one stranded DNA binding replication protein, BALF2, there was no evidence of EBV DNA replication upon activation of the EBV lytic cycle (Fig five). It is consequently achievable that these lytic proteins could not be essential for DNA problems signaling in the pre-replicative stages of EBV but may be important for DNA harm signaling induced for the duration of or immediately after lytic DNA replication.