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ABSTRACT: The EBV latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation, and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including an HDAC inhibitor, TGF-beta cytokine, a demethylating agent (5-Azacytidine), B-cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knock-down of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with Nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53- and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM-dependent. However, over-expression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest ATM enhances Z promoter activity in the context of the intact EBV genome, and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect.
Journal of Virology 09/2012; · 5.40 Impact Factor
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ABSTRACT: The Epstein-Barr virus (EBV) BRRF1 lytic gene product (Na) is encoded within the same immediate-early region as the BZLF1 (Z) and BRLF1(R) gene products, but its role during EBV infection has not been well defined. We previously showed that Na cooperates with the R protein to induce lytic gene expression in latently infected EBV-positive 293 cells, and in some EBV-negative cell lines it can activate the Z promoter in reporter gene assays. Here we show that overexpression of Na alone is sufficient to induce lytic gene expression in several different latently infected epithelial cell lines (Hone-Akata, CNE2-Akata, and AGS-Akata), while knockdown of endogenous Na expression reduces lytic gene expression. Consistent with its ability to interact with tumor necrosis factor receptor-associated factor 2 (TRAF2) in a yeast two-hybrid assay, we demonstrate that Na interacts with TRAF2 in cells. Furthermore, we show that TRAF2 is required for Na induction of lytic gene expression, that Na induces Jun N-terminal protein kinase (JNK) activation in a TRAF2-dependent manner, and that a JNK inhibitor abolishes the ability of Na to disrupt viral latency. Additionally, we show that Na and the tumor suppressor protein p53 cooperate to induce lytic gene expression in epithelial cells (including the C666-1 nasopharyngeal carcinoma cell line), although Na does not appear to affect p53 function. Together these data suggest that Na plays an important role in regulating the switch between latent and lytic infection in epithelial cells and that this effect requires both the TRAF2 and p53 cellular proteins.
Journal of Virology 02/2011; 85(9):4318-29. · 5.40 Impact Factor
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ABSTRACT: The Epstein-Barr virus immediate-early protein, BZLF1 (Z), initiates the switch between latent and lytic infection and plays an essential role in mediating viral replication. Z also inhibits expression of the major receptor for tumor necrosis factor (TNF), TNFR1, thus repressing TNF cytokine signaling, but the mechanism for this effect is unknown. Here, we demonstrate that Z prevents both C/EBPα- and C/EBPβ-mediated activation of the TNFR1 promoter (TNFR1p) by interacting directly with both C/EBP family members. We show that Z interacts directly with C/EBPα and C/EBPβ in vivo and that a Z mutant altered at alanine residue 204 in the bZIP domain is impaired for the ability to interact with both C/EBP proteins. Furthermore, we find that the Z(A204D) mutant is attenuated in the ability to inhibit the TNFR1p but mediates lytic viral reactivation and replication in vitro in 293 cells as well as wild-type Z. Although Z does not bind directly to the TNFR1p in EMSA studies, chromatin immunoprecipitation studies indicate that Z is complexed with this promoter in vivo. The Z(A204D) mutant has reduced interaction with the TNFR1p in vivo but is similar to wild-type Z in its ability to complex with the IL-8 promoter. Finally, we show that the effect of Z on C/EBPα- and C/EBPβ-mediated activation is promoter dependent. These results indicate that Z modulates the effects of C/EBPα and C/EBPβ in a promoter-specific manner and that in some cases (including that of the TNFR1p), Z inhibits C/EBPα- and C/EBPβ-mediated activation.
Journal of Virology 12/2010; 84(23):12362-74. · 5.40 Impact Factor
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ABSTRACT: EBV causes infectious mononucleosis and is associated with certain malignancies. EBV nuclear antigen 1 (EBNA1) mediates EBV genome replication, partition, and transcription, and is essential for persistence of the viral genome in host cells. Here we demonstrate that Hsp90 inhibitors decrease EBNA1 expression and translation, and that this effect requires the Gly-Ala repeat domain of EBNA1. Hsp90 inhibitors induce the death of established, EBV-transformed lymphoblastoid cell lines at doses nontoxic to normal cells, and this effect is substantially reversed when lymphoblastoid cell lines are stably infected with a retrovirus expressing a functional EBNA1 mutant lacking the Gly-Ala repeats. Hsp90 inhibitors prevent EBV transformation of primary B cells, and strongly inhibit the growth of EBV-induced lymphoproliferative disease in SCID mice. These results suggest that Hsp90 inhibitors may be particularly effective for treating EBV-induced diseases requiring the continued presence of the viral genome.
Proceedings of the National Academy of Sciences 02/2010; 107(7):3146-51. · 9.68 Impact Factor
