Involvement of Jun Dimerization Protein 2 (JDP2) in the Maintenance of Epstein-Barr Virus Latency * □S Received for publication, November 2, 2010, and in revised form, April 17, 2011 Published, JBC Papers in Press, April 27, 2011, DOI 10.1074/jbc.M110.199836 Takayuki Murata ‡ , Chieko Noda ‡ , Shinichi Saito ‡ , Daisuke Kawashima ‡ , Atsuko Sugimoto ‡ , Hiroki Isomura ‡ , Teru Kanda ‡ , Kazunari K. Yokoyama § , and Tatsuya Tsurumi ‡1 From the ‡ Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan and the § Center of Excellence for Environmental Medicine, Cancer Center, Graduate Institute of Medicine, Kaohsiung Medical University, 807 Kaohsiung, Taiwan Reactivation of the Epstein-Barr virus from latency is depen- dent on expression of the BZLF1 viral immediate-early protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical inducers such as 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We found that Jun dimerization protein 2 (JDP2) plays a signif- icant role in suppressing Zp activity. Reporter, EMSA, and ChIP assays of a Zp mutant virus revealed JDP2 association with Zp at the ZII cis-element, a binding site for CREB/ATF/AP-1. Sup- pression of Zp activity by JDP2 correlated with HDAC3 associ- ation and reduced levels of histone acetylation. Although introduction of point mutations into the ZII element of the viral genome did not increase the level of BZLF1 production, silencing of endogenous JDP2 gene expression by RNA inter- ference increased the levels of viral early gene products and viral DNA replication. These results indicate that JDP2 plays a role as a repressor of Zp and that its replacement by CREB/ ATF/AP-1 at ZII is crucial to triggering reactivation from latency to lytic replication. The Epstein-Barr virus (EBV) 2 is a human -herpesvirus that predominantly establishes latent infection in B lymphocytes. Only a small percentage of infected cells switch from the latent stage into the lytic cycle to produce progeny viruses. Although the mechanism of EBV reactivation in vivo is not fully under- stood, it is known to be elicited by treatment of latently infected B cells with chemical or biological reagents, such as 12-O-tet- radecanoylphorbol 13-acetate (TPA), calcium ionophore, sodium butyrate, or anti-immunoglobulin, at least in cultured cells. Stimulation of the EBV lytic cascade by any of these leads to expression of two immediate-early genes, BZLF1 and BRLF1. The BZLF1 protein is a transcriptional activator that shares structural similarities to basic leucine zipper (b-Zip) family transcriptional factors and acts as an oriLyt binding protein essential for lytic viral DNA replication. BZLF1 expression alone can trigger the entire reactivation cascade (1–3). Expression of the BZLF1 gene is tightly controlled at the transcriptional level. The BZLF1 promoter (Zp) normally exhibits low basal activity and is activated in response to TPA or the other reagents described above. The minimal sequence of Zp necessary for activation by the inducers is 233 bp in length (4). The region harbors at least three types of cis regulatory elements, referred to as ZI, ZII, and ZIII. Four copies of the ZI element (ZIA–D) are distributed within the minimal Zp. The myocyte enhancer factor 2D binds to ZIA, ZIB, and ZID (5), whereas Sp1 or Sp3 can bind to ZIA, ZIC, and ZID (6). A single ZII element is located near TATA, sharing homology with binding sites for the cyclic AMP-response element-binding protein (CREB), activating transcription factor (ATF), and acti- vator protein-1 (AP-1) family transcriptional factors such as JunB and JunD (7, 8). Two copies of the ZIII element (ZIII-A and -B) bind to the BZLF1 protein. Previous studies have dem- onstrated that both ZI and ZII elements are necessary for the initial activation of the promoter by TPA/ionophore or anti- surface immunoglobulin IgG (2). Then, the expressed BZLF1 protein further activates Zp by binding to ZIIIA and -B (9). BZLF1 also activates transcription of other viral immediate- early or early genes and enhances the lytic infection cycle of the virus. The Jun dimerization protein 2 (JDP2) was initially identified as a binding partner of the AP-1 transcription factor, c-Jun (10). It appears ubiquitously expressed and is involved in a variety of biological phenomena, such as cell differentiation (11–14), apo- ptosis (15, 16), and tumorigenesis (17–22). It can dimerize, through its b-Zip motif, with itself or other b-Zip proteins, such as c-Jun, JunB, JunD, or ATF-2 (10, 11, 23), and function as a general repressor of, at least, AP-1, cAMP-response element, and TPA responsive element-dependent transcription (10, 23). It has been reported that JDP2 recruits histone deacetylase 3 (HDAC3) to the promoters of target genes and inhibits his- tone acetyltransferase activity, thereby suppressing tran- scriptional activity (14, 24). Depending on the context and cell type, however, it can alternatively act as a transcriptional activator (25, 26). In the present study, we obtained evidence that JDP2 sup- presses Zp mainly through interaction with the ZII cis-element. * This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology of Japan 20390137 and 21022055 (to T. T.) and 20790362 and 22790448 (to T. M.), and grants from the Uehara Memorial Research Fund (to T. T.) and the Japan Leukaemia Research Fund (to T. M.). □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. 1 To whom correspondence should be addressed. Tel./Fax: 81-52-764-2979; E-mail: ttsurumi@aichi-cc.jp. 2 The abbreviations used are: EBV, Epstein-Barr virus; Zp, BZLF1 promoter; JDP2, Jun dimerization protein 2; CREB, cyclic AMP-responsive element- binding protein; AP-1, activator protein-1; ATF, activating transcription factor; XBP1(s), spliced form of X-box binding protein 1; TPA, 12-O-tetra- decanoylphorbol13-acetate; b-Zip, basic leucine zipper; CMV, cytomeg- alovirus; HDAC, histone deacetylase; KO, knock-out. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 25, pp. 22007–22016, June 24, 2011 © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. JUNE 24, 2011 • VOLUME 286 • NUMBER 25 JOURNAL OF BIOLOGICAL CHEMISTRY 22007 by guest on June 6, 2020 http://www.jbc.org/ Downloaded from