Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism

The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.     

Phosphatidylinositol 3-kinase is a determinant of responsiveness to B cell antigen receptor-mediated Epstein-Barr virus activation

B cell Ag receptor (BCR) cross-linking with anti-Ig Abs efficiently induces activation of latently infected EBV in some B cell lines, but not in others. The present study was aimed at defining the molecular mechanisms that determine the response to BCR-mediated EBV activation. Comparison of Burkitt's lymphoma-derived Akata, Mutu-I, and Daudi cells, which are representative responders and nonresponders to BCR-mediated EBV activation, respectively, indicated that three signaling pathways, phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK), were activated in anti-Ig-treated Akata and Mutu-I cells. However, in anti-Ig-treated Daudi cells PI3K was not activated, ERK was faintly activated, and p38 MAPK was constitutively phosphorylated irrespective of anti-Ig treatment. Restoration of PI3K activity with insulin-like growth factor 1 restored ERK and p38 MAPK pathways, and was accompanied by EBV activation in anti-Ig-treated Daudi cells. In contrast, a specific inhibitor for PI3K, wortmannin, inhibited EBV activation by anti-Ig Abs in Akata and Mutu-I cells. Transfection assays in EBV-negative Daudi cells revealed that PI3K activated a promoter for BZLF1, which is a switch of EBV activation from a latent infection, in the absence of other EBV products suggesting that the BZLF promoter was a target of BCR signaling, and that PI3K was important for BCR-mediated BZLF1 activation. These results indicate that the absence of PI3K impedes the progression of signals through the BCR and becomes a determinant of unresponsiveness to BCR-mediated EBV activation.     

The ZIIR element of the Epstein-Barr virus BZLF1 promoter plays a central role in establishment and maintenance of viral latency

The Epstein-Barr virus (EBV) BZLF1 gene encodes the immediate-early (IE) protein Zta, which plays a central role in regulating the switch between viral latency and lytic replication. A silencing element, ZIIR, is located between the ZID and ZII positive regulatory elements in the BZLF1 promoter Zp. We report here the phenotypes of variants of EBV strain B95.8 containing base substitution mutations in this ZIIR element. HEK293 cells infected with ZIIR mutant (ZIIRmt) virus produced at least 20-fold more viral IE Zta and Rta and early (E) EAD protein than did cells infected with the parental wild-type (WT) virus, leading to viral DNA replication and production of infectious virus. However, ZIIR mutant virus was 1/10 as efficient as WT virus in establishing proliferating B-cell clones following infection of human primary blood B cells. The ZIIRmt-infected lymphoblastoid cell lines (LCLs) that did grow out exhibited a phenotype similar to the one observed in 293 cells, including marked overproduction of IE and E gene products relative to WT-infected LCLs and lytic replication of the viral genome. Incubation of the ZIIRmt-infected LCLs with the chemical inducer 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to much greater activation of Zp than did the same treatment of WT- or ZVmt-infected LCLs. Furthermore, a protein kinase C (PKC) inhibitor, bis-indolylmaleimide, eliminated this activation by TPA. Thus, we conclude that ZIIR is a potent silencing element of Zp; it plays a key role in establishment and maintenance of EBV latency by inhibiting activation of Zp through the PKC signal transduction pathway.     

Plasma cell-specific transcription factor XBP-1s binds to and transactivates the Epstein-Barr virus BZLF1 promoter

Epstein-Barr virus (EBV) in vivo is known to establish persistent infection in resting, circulating memory B cells and to productively replicate in plasma cells. Until now, the molecular mechanism of how EBV switches from latency to lytic replication in vivo was not known. Here, we report that the plasma cell differentiation factor, XBP-1s, activates the expression of the master regulator of EBV lytic activation, BZLF1. Using reporter assays, we observed that XBP-1s was able to transactivate the BZLF1 promoter, Zp, in a plasma cell line and other lymphoid cell lines but, interestingly, not in epithelial cell lines. We have identified an XBP-1s binding site on the ZID/ZII region of Zp, which when abolished by site-directed mutagenesis led to abrogation of XBP-1s binding and promoter activation. Using the chromatin immunoprecipitation assay, we observed direct binding of XBP-1s to endogenous Zp in an EBV-infected plasma cell line. Finally, in the same cell line, we observed that overexpression of XBP-1s resulted in increased expression of BZLF1, while knockdown of XBP-1s with short hairpin RNA drastically reduces BZLF1 expression. We suggest that EBV harnesses the B-cell terminal differentiation pathway via XBP-1s as a physiological signal to reactivate and begin viral replication. We are currently investigating other signals, such as the endoplasmic reticulum stress response proteins, which act upstream of XBP-1s, to identify other interacting factors that initiate and/or amplify the lytic switch.     

Interplay between PKCδ and Sp1 on histone deacetylase inhibitor-mediated Epstein-Barr virus reactivation

Epstein-Barr virus (EBV) undergoes latent and lytic replication cycles, and its reactivation from latency to lytic replication is initiated by expression of the two viral immediate-early transactivators, Zta and Rta. In vitro, reactivation of EBV can be induced by anti-immunoglobulin, tetradecanoyl phorbol acetate, and histone deacetylase inhibitor (HDACi). We have discovered that protein kinase C delta (PKCδ) is required specifically for EBV reactivation by HDACi. Overexpression of PKCδ is sufficient to induce the activity of the Zta promoter (Zp) but not of the Rta promoter (Rp). Deletion analysis revealed that the ZID element of Zp is important for PKCδ activation. Moreover, the Sp1 putative sequence on ZID is essential for PKCδ-induced Zp activity, and the physiological binding of Sp1 on ZID has been confirmed. After HDACi treatment, activated PKCδ can phosphorylate Sp1 at serine residues and might result in dissociation of the HDAC2 repressor from ZID. HDACi-mediated HDAC2-Sp1 dissociation can be inhibited by the PKCδ inhibitor, Rotterlin. Furthermore, overexpression of HDAC2 can suppress the HDACi-induced Zp activity. Consequently, we hypothesize that HDACi induces PKCδ activation, causing phosphorylation of Sp1, and that the interplay between PKCδ and Sp1 results in the release of HDAC2 repressor from Zp and initiation of Zta expression.     

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 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We 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 increase 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 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.