Involvement of Jun Dimerization Protein 2 (JDP2) in the Maintenance of Epstein-Barr Virus Latency

Reactivation of the Epstein-Barr virus from latency is dependent 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 TPA and calcium ionophore. We here found that Jun dimerization protein 2 (JDP2) plays a significant 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. Suppression of Zp activity by JDP2 correlated with HDAC3 association and reduced levels of histone acetylation. Although introduction of point mutations into the ZII element of the viral genome did not the level of BZLF1 production, silencing of endogenous JDP2 gene expression by RNA interference increased the levels of viral early gene products and viral DNA replication. These results indicate that JDP2 plays a role as

2 enhancer factor 2D (MEF2D) binds to ZIA, ZIB and ZID (5), while 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 activator protein-1 (AP-1) family transcriptional factor such as Jun-B and Jun-D (7,8). Two copies of the ZIII element 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.
In the present study, we obtained evidence that JDP2 suppresses Zp mainly through interaction with the ZII cis-element. The lytic life cycle of the virus was found to be significantly enhanced by silencing of endogenous JDP2 expression. We also found JDP2 supported recruitment of HDAC3 to Zp. These results indicate that JDP2 plays critical roles in regulation of the latent-lytic switch in EBV infection.

Genetic manipulation of EBV-BAC DNA and cloning of HEK293 cells with EBV-BAC -EBV-BAC
DNA was provided by W. Hammerschmidt (34).
Homologous recombination was carried out in E. coli as described previously (28).
To prepare an mZII mutant of EBV-BAC, a transfer DNA fragment for the first recombination was  6 Therefore, we here adopted siRNA technology, using synthetic oligonucleotides that form duplex RNA encoding partial nucleotides from JDP2. As shown in Fig The site-directed mutation of the ZIII element, which prevents BZLF1 binding, failed to respond to ectopic expression of BZLF1 protein (Fig. 1D) as expected, but its promoter activity was increased by si-JDP2 treatment (Fig. 1D, pZpmZIII-  Therefore, JDP2 binds to ZII, but not to ZIIIA or ZIIIB cis-element of Zp.

Association of JDP2 with the BZLF1 promoter in vivo.
To further verify the reporter assays and EMSA results, we performed ChIP analysis using cells containing wild-type or BZLF1-knockout (BZLF1KO) EBV-BAC (27). We here used BZLF1KO mutant strain, in order to exclude the possibility that BZLF1 is involved in the association between JDP2 and the BZLF1 promoter. The BZLF1 promoter region was obviously co-precipitated with Flag-tagged JDP2 (Fig. S1A) (Fig. 3A). Thus, the association proved to be intact, indicating that BZLF1 is not needed for JDP2 binding to the BZLF1 promoter sequence.
Since the above documented results indicated JDP2 acts to suppress the BZLF1 promoter by binding to the ZII cis-element, we generated recombinant EBV with a point mutation in the ZII element (Fig. 4A) as the pZpmZII-luc vector (Fig. 1), and that the EBV-BAC mZII/R had the same sequence as the wild-type virus, as intended. Integrity of the BAC DNA was checked by BamHI digestion followed by electrophoresis to confirm that the recombinant viruses did not carry obvious deletions or insertions (Fig. 4B).
The BamHI-Z fragment was too short to be clearly detected.
Recombinant EBV-BAC DNA was introduced into The competitive nature of JDP2 with CREB was reconfirmed and quantified in Fig. 5. When JDP2 was knocked down, Flag-tagged CREB association with the Zp was significantly higher than that with the control siRNA (Fig. 5A). Immunoblotting data indicated the expression level of Flag-CREB was not so affected by si-JDP2 (Fig. 5C). These data indicate that JDP2 inhibits transcriptional activity of Zp by competitively occupying the ZII binding site.
We then examined whether silencing of JDP2 influence viral lytic replication another cell line (Fig. S4).
Akata cells featuring latent infection were treated with the siRNA against JDP2. The levels of BZLF1 protein and early proteins, such as BMRF1, BALF5, and BALF2, were also enhanced, especially when treatment was combined with exposure to anti-IgG (Fig. S4).
Expression of dJDP2 mutant served as a negative control, as it lacks its b-Zip domain, and thus be not functional. In suppressing transcriptional activity (14,24). As exhibited previously, exogenously expressed Flag-tagged JDP2 was able to associate with Zp (Fig. 7A, left part), whereas its b-Zip deletion mutant lost the ability (Fig. 7A, right part). Association of JDP2 with the BZLF1 promoter correlated with increased HDAC3 recruitment to the promoter (Fig. 7A). This HDAC3 recruitment by JDP2 correlated to reduced levels of histone H3 acetylation (Fig.   7B). To extend these results, endogenous JDP2 was knocked down by siRNA (Fig. 7C). Recruitment of endogenous HDAC3 to Zp was weak although appreciable in the si-control case, whereas treatment 9 with si-JDP2 decreased the HDAC3 interaction with Zp ( Fig. 7C). The reduction in HDAC3 recruitment by si-JDP2 caused increment in histone H3 acetylation levels (Fig. 7D). Furthermore, JDP2 association with HDAC3 was confirmed by immunoprecipitation (Fig.   7E). These results suggest that HDAC3 recruitment to Zp, which coincides with lower levels of acetylated histone H3, is efficiently mediated by JDP2 protein.

DISCUSSION
In this report, we document evidence that JDP2 is For example, BZLF1 expression is highly leaky in AGS cells infected with EBV, at least partly due to abundant c-Jun and its binding to the ZII element (36). We are confident that JDP2 acts negatively on EBV reactivation, because knockdown of the gene substantially enhanced expression of viral immediate early, early genes and viral DNA replication (Fig. 6,S4).
In Fig. 6, huge amount (1,000-fold or even more of endogenous level of JDP2) of forced expression of JDP2 caused only 50% reduction in BZLF1 mRNA levels.
One might argue that this reduction must have been more extensive. We speculate that the ZII motif of the promoter might already be occupied by endogenous b-Zip transcription factors, including JDP2, and thus exogenouly supplied JDP2 could not replace effectively.
Otherwise, JDP2 might be just one of several proteins that serve to restrict the BZLF1 expression, and the restriction by JDP2 could easily be achieved by the presence of relatively small amount of the protein.
Since JDP2 and viral BZLF1 are both b-Zip type transcriptional factors, we tested if they could interact with each other. Immunoprecipitation assays clearly demonstrated that JDP2 and BZLF1 associated through their b-Zip elements (data not shown), and we thus examined if the factors could act cooperatively on the BZLF1 promoter (Fig. 1). However, we observed no evidence of such cooperation, and the ZIII cis-element, the binding motif for BZLF1, did not appear to be involved in the suppression by JDP2 (Fig. 1).