The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection.

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells.

b Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas, BARF1 expression is restricted to the viral lytic replication cycle. In this report, the transcriptional regulation of BARF1 during lytic replication is unraveled. Bisulfite sequencing of various cell lines indicated a high level of methylation of the BARF1 gene control region. A BARF1 promoter luciferase reporter construct was created using a CpG-free vector, enabling true assessment of promoter methylation. Induction of the EBV lytic cycle is mediated by the immediate-early proteins BZLF1 (Z) and BRLF1 (R). R was found to activate expression of the BARF1 promoter up to 250-fold independently of Z and unaffected by BARF1 promoter methylation. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and specific mutagenesis of the R-responsive elements (RREs) demonstrated direct binding of R to RREs between nucleotides ؊554 and ؊327 relative to the BARF1 transcriptional ATG start site. The kinetics of BARF1 expression upon transactivation by R showed that BARF1 mRNA was expressed within 6 h in the context of the viral genome. In conclusion, expression of the BARF1 protein during lytic replication is regulated by direct binding of R to multiple RREs in the gene control region and is independent of the promoter methylation status. The early kinetics of BARF1 upon transactivation by R confirm its status as an early gene and emphasize the necessity of early immune modulation during lytic reactivation.

The Epstein-Barr virus (EBV) latent-to-lytic switch is an essential part of the viral life cycle, but the cellular factors that promote viral reactivation are not well defined. In this report, we demonstrate that the cellular transcription factor Oct-1 cooperates with the EBV immediate-early protein BRLF1 (R, Rta) to induce lytic viral reactivation. We show that cotransfected Oct-1 enhances the ability of BRLF1 to activate lytic gene expression in 293 cells stably infected with a BRLF1-defective EBV mutant (BRLF1-stop) and that Oct-1 increases BRLF1-mediated activation of lytic EBV promoters in reporter gene assays. We find that Oct-1 interacts directly with BRLF1 in vitro and that a mutant BRLF1 protein (the M140A mutant) attenuated for the ability to interact with Oct-1 in vitro is also resistant to Oct-1-mediated transcriptional enhancement in 293 BRLF1-stop cells. Furthermore, we show that cotransfected Oct-1 augments BRLF1 binding to a variety of lytic EBV promoters in chromatin immunoprecipitation (ChIP) assays (including the BZLF1, BMRF1, and SM promoters) and that BRLF1 tethers Oct-1 to lytic EBV promoters. In addition, we demonstrate that an Oct-1 mutant defective in DNA binding (the S335D mutant) still retains the ability to enhance BRLF1 transcriptional effects. Finally, we show that knockdown of endogenous Oct-1 expression reduces the level of constitutive lytic EBV gene expression in both EBV-positive B-cell and EBV-positive epithelial cell lines. These results suggest that Oct-1 acts as a positive regulator of EBV lytic gene expression and that this effect is at least partially mediated through its interaction with the viral protein BRLF1. Epstein-Barr virus (EBV), or human herpesvirus 4 (HHV4), is a double-stranded DNA gammaherpesvirus which infects approximately 90% of the world's population (66). It causes a relatively mild primary disease if acquired early in life and infectious mononucleosis if acquired after adolescence. EBV is also associated with several B-cell and epithelial cell cancers, including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma (NPC), and gastric carcinoma (66, 101).EBV infection persists for the life of the host as a chronic asymptomatic infection by establishing latency in memory B cells (77). However, to be transmitted from host to host, and from cell to cell, the virus periodically reactivates from latency and converts to the lytic form of replication (66). Reactivation of lytic EBV infection in host cells is controlled at the level of the BZLF1 (Z, Zta, ZEBRA, EB1) and BRLF1 (R, Rta) viral promoters, as well as the activity of the BZLF1 and BRLF1 gene products (24,42,95). The BZLF1 and BRLF1 genes encode transcription factors that cooperatively turn on the expression of the early lytic viral genes involved in lytic viral replication (2,9,16,17,23,24,26,29,31,41,50,62,68), and the overexpression of either BZLF1 or BRLF1 is sufficient to induce lytic gene expression in many latently infected cell lines (13-15, 63, 83, 96). Since the combination of both BZL...

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/EBP␣-and C/EBP␤-mediated activation of the TNFR1 promoter (TNFR1p) by interacting directly with both C/EBP family members. We show that Z interacts directly with C/EBP␣ and C/EBP␤ 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/EBP␣-and C/EBP␤-mediated activation is promoter dependent. These results indicate that Z modulates the effects of C/EBP␣ and C/EBP␤ in a promoter-specific manner and that in some cases (including that of the TNFR1p), Z inhibits C/EBP␣-and C/EBP␤-mediated activation.