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 (PKC␦) is required specifically for EBV reactivation by HDACi. Overexpression of PKC␦ 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 PKC␦ activation. Moreover, the Sp1 putative sequence on ZID is essential for PKC␦-induced Zp activity, and the physiological binding of Sp1 on ZID has been confirmed. After HDACi treatment, activated PKC␦ 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 PKC␦ inhibitor, Rotterlin. Furthermore, overexpression of HDAC2 can suppress the HDACi-induced Zp activity. Consequently, we hypothesize that HDACi induces PKC␦ activation, causing phosphorylation of Sp1, and that the interplay between PKC␦ and Sp1 results in the release of HDAC2 repressor from Zp and initiation of Zta expression.Epstein-Barr virus (EBV), a human oncogenic virus, infects two types of human cells predominantly, B lymphocytes and epithelial cells, and EBV infection also is associated with an array of malignancies derived from these two cell types, including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and gastric carcinoma (49). Being a human gammaherpesvirus, EBV undergoes two cycles: latency and lytic replication. Although most latent products are important for its ability to immortalize cells, and the virus is present in a latent form in most EBV-associated cancer tissues, serological studies revealed that elevated antibody titers against some lytic antigens, or increased viral DNA load in serum, are risk factors for tumor development (8,33). In addition, experiments using the SCID mouse model suggested that Zta, an EBV lytic transactivator, is crucial for tumor formation (43). In vitro, experimental approaches also revealed that EBV lytic products may contribute to the pathogenesis of EBV-associated diseases, via upregulation of cytokines, cell growth factors or antiapoptotic proteins (5, 48, 50). Thus, the reactivation of EBV not only leads to the production of viral progeny but also facilitates viral pathogenesis.In vitro, EBV persists predominantly in a latent form in host cells. However, lytic replication can be elicited conditionally by many different chemicals and physical stimuli or by ectopic transfection with two transactivators, Zta or Rta (49). These inducing agents provide the best available models for the study of essential factors involved in the switch of EBV from latency to lytic replication. Tetradecanoyl phorbol acetate (TPA) and anti-Ig are...
Epstein-Barr virus (EBV), an oncogenic herpesvirus, has the potential to immortalize primary B cells into lymphoblastoid cell lines (LCLs) in vitro.During immortalization, several EBV products induce cytokines or chemokines, and most of these are required for the proliferation of LCLs. Interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, is upregulated after EBV infection, and this upregulation is detectable in all LCLs tested. EBV latent membrane protein 1 (LMP1) is responsible for inducing IL-32 expression at the mRNA and protein levels. Mechanistically, we showed that this LMP1 induction is provided by the p65 subunit of NF-B, which binds to and activates the IL-32 promoter. Furthermore, the short hairpin RNA (shRNA)-mediated depletion of endogenous LMP1 and p65 in LCLs suppressed IL-32 expression, further suggesting that LMP1 is the key factor that stimulates IL-32 in LCLs via the NF-B p65 pathway. Functionally, knockdown of IL-32 in LCLs elicits viral reactivation and affects cytokine expression, but it has no impact on cell proliferation and apoptosis. Of note, we reveal the mechanism whereby IL-32 is involved in the maintenance of EBV viral latency by inactivation of Zta promoter activity. This atypical cytoplasmic IL-32 hijacks the Zta activator protein kinase C␦ (PKC␦) and inhibits its translocation from the cytoplasm to the nucleus, where PKC␦ binds to the Zta promoter and activates lytic cycle progression. These novel findings reveal that IL-32 is involved in the maintenance of EBV latency in LCLs. This finding may provide new information to explain how EBV maintains latency, in addition to viral chromatin structure and epigenetic modification. IMPORTANCEEBV persists in two states, latency and lytic replication, which is a unique characteristic of human infections. So far, little is known about how herpesviruses maintain latency in particular tissues or cell types. EBV is an excellent model to study this question because more than 90% of people are latently infected. EBV can immortalize primary B cells into lymphoblastoid cell lines in vitro. Expression of IL-32, a novel atypical cytoplasmic proinflammatory cytokine, increased after infection. The expression of IL-32 was controlled by LMP1. In investigating the regulatory mechanism, we demonstrated that the p65 subunit of NF-B is required for this upregulation. Of note, the important biological activity of IL-32 was to trap protein kinase C␦ in the cytoplasm and prevent it from binding to the Zta promoter, which is the key event for EBV reaction. So, the expression of LMP1-induced IL-32 plays a role in the maintenance of EBV latency. E pstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus: more than 90% of the human population are latently infected (1). Of note, EBV has been reported to be associated with Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), natural killer (NK)/T-cell lymphoma, AIDS-associated lymphoma, and posttransplantation lymphoproliferative disorder (PTLD) (2). The strongest evidence for EBV onco...
Key Points• EBV LMP2A alters B-cell gene expression; E47 and PU.1 are repressed by LMP2A, resulting in downregulation of MHC class II expression.Oncogenic Epstein-Barr virus (EBV) uses various approaches to escape host immune responses and persist in B cells. Such persistent infections may provide the opportunity for this virus to initiate tumor formation. Using EBV-immortalized lymphoblastoid cell lines (LCLs) as a model, we found that the expression of major histocompatibility complex (MHC) class II and CD74 in B cells is repressed after EBV infection. Class II transactivator (CIITA) is the master regulator of MHC class II-related genes. As expected, CIITA was downregulated in LCLs. We showed that downregulation of CIITA is caused by EBV latent membrane protein 2A (LMP2A) and driven by the CIITA-PIII promoter. Furthermore, we demonstrated that LMP2A-mediated E47 and PU.1 reduction resulted in CIITA suppression. Mechanistically, the LMP2A immunoreceptor tyrosine-based activation motif was critical for the repression of E47 and PU.1 promoter activity via Syk, Src, and the phosphatidylinositol 3-kinase/Akt pathway. Elimination of LMP2A in LCLs using a shLMP2A approach showed that the expression levels of E47, PU.1, CIITA, MHC class II, and CD74 are reversed. These data indicated that the LMP2A may reduce MHC class II expression through interference with the E47/PU.1-CIITA pathway. Finally, we demonstrated that MHC class II may be detected in tonsils and EBV-negative Hodgkin disease but not in EBV-associated posttransplant lymphoproliferative disease and Hodgkin disease. (Blood. 2015;125(14):2228-2238) IntroductionDuring infection, viruses face the various challenges of the host immune response and have evolved a number of immune evasion strategies to enable successful infection of the host cells. Epstein-Barr virus (EBV) is a ubiquitous, human g-herpesvirus that persistently infects more than 90% of the human population.1 EBV has evolved several mechanisms to escape immune surveillance: EBV limits the expression of its proteins and persists in immune B cells. EBV uses various strategies to attenuate the first line of innate immunity; for example, latent membrane protein 1 (LMP1) negatively regulates the expression of the important sensor Toll-like receptor 9, 2 B-cell receptor F1 (BCRF1)-encoded viral interleukin-10 inhibits interferon production, 3 and the tegument protein BPLF1 blocks Toll-like receptor signaling. 4 In counteracting the adaptive immune response, EBV interferes with the major histocompatibility complex (MHC) class I and II antigen presentations, which are the key factors for adaptive immunity. MHC antigens are critical in the cellular immune response, which is important for viral clearance. Several studies have addressed the question how EBV downregulates MHC class I antigen expression. EBNA1 has a glycinealanine repeat motif that protects it from degradation and also inhibits its own synthesis. 5,6 The EBV lytic protein BNLF2a interferes with both the peptide and adenosine triphosphate binding ...
e Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.
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