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doi:10.1371/journal.ppat.1001114. not directly bind the nucleotide (nt) ?660 Rp BRE. siRNA knockdown of BLIMP1 inhibited 12-and gene) and R (also called Rta or EB2, the product of the gene), respectively (13). In most EBV+ cell lines, synthesis of Z is sufficient to initiate reactivation (14,C17). R can frequently initiate reactivation as well, given that R and Z are transcription factors that can usually activate each other’s promoters (18,C24). However, R, not Z, is necessary in some MYO9B cases such as telomerase-immortalized normal oral keratinocytes (NOK) that have been infected with the Akata strain of EBV (25). Together, R and Z induce expression of the viral early (E) genes, including BMRF1, which encodes the viral DNA polymerase processivity factor (also known as early antigen diffuse [EAD]) (26,C29). Thus, regulation of Zp and Rp serves as the gatekeeper to the EBV latent-to-lytic switch in a cell-type-dependent manner. Zp has been intensively studied, with there being numerous well-mapped positive and negative regulatory domains (reviewed in references 30 to 33). Unfortunately, much less is known to date about regulation of Rp (reviewed in reference 33) (see Fig. 4A below). Among Rp’s known (46,C49). BLIMP1 represses its target genes through a variety of mechanisms, including recruiting Groucho family proteins, histone deacetylase 1 and 2 (HDAC1/2), and histone methyltransferase G9a (50,C54). Open in a separate window FIG 5 PR and zinc finger domains of BLIMP1 are critical for inducing reactivation. (A) Schematic showing structures of BLIMP1 wild-type and variants with deletions in the PRDI-BF1-RIZ1 (PR), proline-rich (Pro), and Zn finger DNA-binding (Zn) domains. The figure is not drawn to scale. (B) Immunoblot showing relative levels of some EBV lytic proteins following expression of WT BLIMP1 and the BLIMP1 variants depicted in panel A. HONE-Akata cells in 35-mm-diameter wells were transfected with (i) 300 ng of the indicated pcDNA3-BLIMP1 variant or pcDNA3.1 and (ii) 700 ng ML 7 hydrochloride of pSG5. Whole-cell extracts were prepared 48 h posttransfection. All samples were present in the same immunoblots, with irrelevant lanes omitted. GAPDH served as a loading control. (C) Luciferase assays showing relative abilities of the variant BLIMP1 proteins to activate Rp. AGS cells in 22-mm-diameter wells were cotransfected with (i) 450 ng of pCpGL-Rp-673 and (ii) 50 ng of the indicated pcDNA3-BLIMP1 variant depicted in panel A or pcDNA3.1. Data were ML 7 hydrochloride normalized to WT BLIMP1-induced activation of Rp-673. Values are representative means of assays performed in triplicate on two separate occasions. Error bars indicate standard deviations within one representative set of assays performed in triplicate. Given that the PAX5 and Oct-2 proteins inhibit Z activity (55, 56) and that PAX5 represses synthesis of XBP-1 in lymphoid cells (57), one might expect that addition of BLIMP1 would induce EBV lytic gene expression in some EBV+ B-cell lines under some conditions; this hypothesis has been validated (58). Other investigators ML 7 hydrochloride reported that while expression of EBV Z protein was restricted to BLIMP1-positive epithelial cells in oral hairy leukoplakia (OHL) tissue samples, this relationship did not ML 7 hydrochloride hold in IM tonsillar B cells, suggesting that BLIMP1 may be necessary for induction of EBV lytic gene expression during differentiation of epithelial but not B cells (59). Here, we show that expression of BLIMP1 is sufficient to induce EBV lytic gene expression in EBV+ epithelial cell lines derived from gastric cancers, NPCs, and NOK. We demonstrate that BLIMP1 is also necessary for differentiation-induced lytic reactivation of EBV in at least some epithelial cell types. We further show that BLIMP1 can induce transcriptional activation of Rp ML 7 hydrochloride over 300-fold as well as Zp at 20- to 50-fold in some epithelial cell lines, and we identify several regions within Rp that act synergistically to enable this high-level activation by BLIMP1. Thus, we conclude that BLIMP1 is a key player in EBV lytic reactivation in epithelial cells; BLIMP1 does so, at least in part, by inducing expression of the as well as the gene. MATERIALS AND METHODS Cell lines. The gastric carcinoma cell line AGS (obtained from the American Type Culture Collection [ATCC]) and the EBV-infected clone derived from it, AGS-Akata (a gift from Lindsey Hutt-Fletcher), were maintained in F12 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 100 U/ml penicillin and 100 g/ml streptomycin (Pen-Strep; Life Technologies). The NPC cell line HONE1 (a gift from Ron Glaser), the EBV-infected clone derived from it, HONE-Akata (a gift from Lawrence Young), the EBV-infected NPC.