Epstein-Barr Virus Lytic Reactivation Research Articles

Sickle cell trait is not associated with endemic Burkitt lymphoma: An ethnicity and malaria endemicity‐matched case–control study suggests factors controlling EBV may serve as a predictive biomarker for this pediatric cancer

Endemic Burkitt lymphoma (eBL) is associated with Epstein–Barr virus (EBV) and Plasmodium falciparum coinfections. Malaria appears to dysregulate immunity that would otherwise control EBV, thereby contributing to eBL etiology. Juxtaposed to human genetic variants associated with protection from malaria, it has been hypothesized that such variants could decrease eBL susceptibility, historically referred to as “the protective hypothesis.” Past studies attempting to link sickle cell trait (HbAS), which is known to be protective against malaria, with protection from eBL were contradictory and underpowered. Therefore, using a case–control study design, we examined HbAS frequency in 306 Kenyan children diagnosed with eBL compared to 537 geographically defined and ethnically matched controls. We found 23.8% HbAS for eBL patients, which was not significantly different compared to 27.0% HbAS for controls [odds ratio (OR) = 0.85; 95% confidence interval (CI) 0.61–1.17; p-value = 0.33]. Even though cellular EBV titers, indicative of the number of latently infected B cells, were significantly higher (p-value < 0.0003) in children residing in malaria holoendemic compared to hypoendemic areas, levels were not associated with HbAS genotype. Combined, this suggests that although HbAS protects against severe malaria and hyperparasitemia, it is not associated with viral control or eBL protection. However, based on receiver operating characteristic curves factors that enable the establishment of EBV persistence, in contrast to those involved in EBV lytic reactivation, may have utility as an eBL precursor biomarker. This has implications for future human genetic association studies to consider variants influencing control over EBV in addition to malaria as risk factors for eBL.What's new?Although the hypothesis dates back to 1970, studies of the “protective effect” of sickle cell trait (HbAS) on the development of endemic Burkitt lymphoma (eBL) have led to contradictory conclusions. This new study analyzed an unprecedented number of eBL cases and highlighted the importance of matching controls on ethnicity as well as malaria endemicity. The findings contribute to resolving the controversy by showing that HbAS frequency does not differ between children diagnosed with eBL and healthy children. The study also examined cellular Epstein-Barr Virus (EBV) titers, which in contrast to plasma EBV levels may serve as an informative eBL biomarker.

Viral Genome Methylation Differentially Affects the Ability of BZLF1 versus BRLF1 To Activate Epstein-Barr Virus Lytic Gene Expression and Viral Replication

The Epstein-Barr virus (EBV) immediate-early proteins BZLF1 and BRLF1 can both induce lytic EBV reactivation when overexpressed in latently infected cells. Although EBV genome methylation is required for BZLF1-mediated activation of lytic gene expression, the effect of viral genome methylation on BRLF1-mediated viral reactivation has not been well studied. Here, we have compared the effect of viral DNA methylation on BZLF1- versus BRLF1-mediated activation of lytic EBV gene transcription and viral genome replication. We show that most early lytic viral promoters are preferentially activated by BZLF1 in the methylated form, while methylation decreases the ability of BRLF1 to activate most early lytic promoters, as well as the BLRF2 late viral promoter. Moreover, methylation of bacmid constructs containing the EBV genome enhances BZLF1-mediated, but decreases BRLF1-mediated, early lytic gene expression. Methylation of viral promoter DNA does not affect BRLF1 binding to a variety of different CpG-containing BRLF1 binding motifs (RREs) in vitro or in vivo. However, BRLF1 preferentially induces H3K9 histone acetylation of unmethylated promoters in vivo. The methylated and unmethylated forms of an oriLyt-containing plasmid replicate with similar efficiency when transfected into EBV-positive cells that express the essential viral replication proteins in trans. Most importantly, we demonstrate that lytic viral gene expression and replication can be induced by BRLF1, but not BZLF1, expression in an EBV-positive telomerase-immortalized epithelial cell line (NOKs-Akata) in which lytic viral gene promoters remain largely unmethylated. These results suggest that the unmethylated form of the EBV genome can undergo viral reactivation and replication in a BRLF1-dependent manner.

The cellular ataxia telangiectasia-mutated kinase promotes epstein-barr virus lytic reactivation in response to multiple different types of lytic reactivation-inducing stimuli.

The Epstein-Barr virus (EBV) latent-to-lytic switch is mediated by the viral proteins BZLF1 (Z), BRLF1 (R), and BRRF1 (Na). Since we previously showed that DNA-damaging agents (including chemotherapy and irradiation) can induce EBV lytic reactivation and recently demonstrated that wild-type p53 contributes to lytic reactivation, we investigated the role of the ATM kinase during EBV reactivation. ATM phosphorylates and activates p53, as well as numerous other substrates involved in the cellular DNA damage response. Using an ATM inhibitor (KU55933), we found that ATM activity is required for efficient induction of EBV lytic gene expression by a variety of different stimuli, including a histone deacetylase (HDAC) inhibitor, the transforming growth factor β (TGF-β) cytokine, a demethylating agent (5-azacytidine), B cell receptor engagement with anti-IgG antibody, hydrogen peroxide, and the proteosome inhibitor bortezomib. In EBV-infected AGS (gastric) cells, knockdown of ATM, or p53, expression inhibits EBV reactivation. Conversely, treatment of these cells with nutlin-3 (which activates p53 and ATM) robustly induces lytic reactivation in a p53- and ATM-dependent manner. The ability of the EBV R and Na proteins to induce lytic reactivation in EBV-infected AGS cells is ATM dependent. However, overexpression of Z induces lytic gene expression in the presence or absence of ATM activity. Our results suggest that ATM enhances Z promoter activity in the context of the intact EBV genome and that p53 contributes to the ATM effect. Nevertheless, since we found that ATM inhibitors also reduce lytic reactivation in Burkitt lymphoma cells that have no p53, additional ATM substrates must also contribute to the ATM effect.

Signal transducer and activator of transcription 3 (STAT3) controls susceptibility to Epstein-Barr virus reactivation in B cells

Epstein-Barr Virus (EBV) is known to cause malignancies in immunocompromised individuals, including lymphomas and lymphoproliferative disorders. Reactivation of EBV from latency is important in the pathogenesis of such malignancies. Therapeutically, oncolysis of tumors harboring lytic EBV holds promise. However, exposure of latently infected cultured B cells to lytic cycle inducing stimuli results in virus reactivation within only 20% to 50% of cells. Host cell determinants, that govern this susceptibility to lytic reactivat ion within latently infected B cells, are not well understood. Our research has previously identified that higher levels of signal transducer and activator of transcription 3 (STAT3) within cells correlate with resistance to EBV lytic reactivation. We now investigate whether inhibiting function of STAT3 promotes susceptibility of latently infected B cells to known lytic cycle inducing stimuli or results in induction of EBV lytic cycle. We found that pharmacological inhibition of STAT3 phosphorylation by Janus Kinase 2 (JAK2) inhibitors (AG490 or WP1066) in latently infected cells (HH514-16 Burkitt lymphoma and B95-8 lymphoblastoid cells) increased lytic reactivation by chemical stimuli that function by distinct mechanisms. Moreover, functional inhibition of STAT3 alone resulted in lytic reactivation in these cells. EBV-Lymphoblastoid cell lines (LCL) newly generated from healthy individuals also demonstrated increased susceptibility to lytic cycle inducing stimuli in the presence of AG490 but variable susceptibility to lytic reactivation when exposed to AG490 alone. Since this lack of uniform response to AG490 alone could be due to inadequate functional suppression of STAT3, we examined EBV-LCLs derived from patients with Autosomal Dominant Hyperimmunoglobulin-E syndrome (AD-HIES or Job syndrome). Patients with AD-HIES carry a dominant negative mutation in their Stat3 gene resulting in lower basal levels of functional STAT3. When LCLs from AD-HIES patients were exposed to a JAK2 inhibitor alone, we observed a strong increase in lytic reactivation by expression of early and late lytic antigens over LCLs derived from healthy individuals. Lytic reactivation in the presence of AG490 occurred in these cells despite lack of discernable increase from basal levels of expression of ZEBRA, the viral lytic switch protein, when compared to cells not exposed to AG490. Thus, STAT3 is important in determining susceptibility to EBV reactivation. Fully understanding how STAT3 governs such susceptibility can lead to novel therapeutic strategies for EBV-related diseases.

Epstein-Barr virus lytic gene expression is tightly linked to ER stress but not cytotoxicity with bortezomib or nelfinavir

Epstein-Barr virus (EBV) is associated with AIDS-related lymphomas and other malignancies. We have previously shown that the proteasome inhibitor bortezomib is an activator of EBV lytic gene expression and that these effects are mediated by ER stress and the unfolded protein response (UPR) [1]. We investigated the relationship between the induction of UPR and EBV lytic gene expression with a variety of UPR inducers, as well as the association of their viral and antitumor effects in a variety of tumor cell lines. Bortezomib, thapsigargin, and tunicamycin activate the UPR and EBV lytic cycle. Recently, nelfinavir has also been reported to lead to ER-stress and the UPR. We found a dose-dependent relationship with bortezomib and with nelfinavir for the induction of the UPR markers (Bip, ATF4, XBP1s, and CHOP10), EBV lytic gene expression (measured as ZTA RNA), and cell toxicity in Burkitt’ sl ymphoma cell lines.. Blocking ER stress and UPR activation, by cycloheximide (CHX) treatment or by Bip knockdown, diminished ZTA induction but had no effect on cellular toxicity. We also studied EBV lymphoblastoid cell lines (LCLs). In contrast to the BL cell lines, bortezomib did not induce ER stress, activate the UPR or lead to EBV lytic gene expression but was nonetheless toxic to LCLs. These results indicate that bortezomib and nelfinavir both induce ER stress and UPR leading to EBV lytic reactivation in BL cells. UPR induction corresponds with EBV lytic gene induction but appears to be distinct from cellular toxicity. Our findings suggest that ER stress, UPR and viral activation are closely linked but may be separable from the cytotoxic effects of some pharmacologic inducers. Bortezomib and nelfinavir may serve as laboratory and clinical tools for manipulating viral gene expression in EBV associated malignancies.

Functions of the Epstein-Barr Virus EBNA1 Protein in Viral Reactivation and Lytic Infection

EBNA1 is the only nuclear Epstein-Barr virus (EBV) protein expressed in both latent and lytic modes of infection. While EBNA1 is known to play several important roles in latent infection, the reason for its continued expression in lytic infection is unknown. Here we identified two roles for EBNA1 in the reactivation of latent EBV to the lytic cycle in epithelial cells. First, EBNA1 depletion in latently infected cells was shown to positively contribute to spontaneous EBV reactivation, showing that EBNA1 has a role in suppressing reactivation. Second, when the lytic cycle was induced, EBNA1 depletion decreased lytic gene expression and DNA amplification, showing that it positively contributed to lytic infection. Since we have previously shown that EBNA1 disrupts promyelocytic leukemia (PML) nuclear bodies, we investigated whether this function could account for the effects of EBNA1 on lytic infection by repeating the experiments with cells lacking PML proteins. In the absence of PML, EBNA1 did not promote lytic infection, indicating that the EBNA1-mediated PML disruption is responsible for promoting lytic infection. In keeping with this conclusion, PML silencing was found to be sufficient to induce the EBV lytic cycle. Finally, by generating cells with single PML isoforms, we showed that individual PML isoforms were sufficient to suppress EBV lytic reactivation, although PML isoform IV (PML IV) was ineffective because it was most efficiently degraded by EBNA1. Our results provide the first function for EBNA1 in lytic infection and show that EBNA1 interactions with PML IV lead to a loss of PML nuclear bodies (NBs) that promotes lytic infection.

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells.

The company malaria keeps

Co-infection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) are implicated in the cause of endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in equatorial Africa. Although the causal association between EBV and eBL has been established, P. falciparum malaria's role is not as clearly defined. This review focuses on how malaria may disrupt EBV persistence and immunity. Two mutually compatible theories have been proposed. One suggests that P. falciparum malaria induces polyclonal B-cell expansion and lytic EBV reactivation, leading to the expansion of latently infected B cells and the likelihood of a c-myc translocation, a hallmark of Burkitt lymphoma tumors. The other advocates that EBV-specific T-cell immunity is impaired during P. falciparum malaria co-infection, either as a cause or consequence of enhanced EBV replication, leading to loss of viral control. Advancements in our ability to query the complexity of human responses to infectious diseases have stimulated interest in eBL pathogenesis. EBV is necessary but not sufficient to cause eBL. A more dynamic model encompasses incremental contributions from both chronic and acute P. falciparum malaria leading to alterations in EBV persistence and EBV-specific immunity that culminate in eBL. A better understanding of how P. falciparum malaria modifies EBV infections in children may allow us to anticipate reductions in eBL incidence coinciding with malaria control programs.

Transforming Growth Factor β-Induced Reactivation of Epstein-Barr Virus Involves Multiple Smad-Binding Elements Cooperatively Activating Expression of the Latent-Lytic SwitchBZLF1Gene

Transforming growth factor β (TGF-β) physiologically induces Epstein-Barr virus (EBV) lytic infection by activating the expression of EBV's latent-lytic switch BZLF1 gene. Liang et al. (J. Biol. Chem. 277:23345-23357, 2002) previously identified a Smad-binding element (SBE) within the BZLF1 promoter, Zp; however, it accounts for only 20 to 30% of TGF-β-mediated activation of transcription from Zp. Here, we identified additional factors responsible for the rest of this activation. The incubation of EBV-positive MutuI cells with a TGF-β neutralizing antibody or inhibitors of the TGF-β type I receptor (TβRI) or Smad3 eliminated the TGF-β-induced reactivation of EBV. The coexpression of Smad2, Smad3, and Smad4 together with a constitutively active form of TβRI induced 15- to 25-fold transcription from Zp in gastric carcinoma AGS cells. By electrophoretic mobility shift assays, we identified four additional Smad-binding elements, named SBE2 to SBE5. Substitution mutations in individual SBEs reduced Smad-mediated activation of Zp by 20 to 60%; together, these mutations essentially eliminated it. Chromatin immunoprecipitation assays confirmed that Smad4 newly bound the Zp region of the EBV genome following the incubation of MutuI cells with TGF-β. SBE2 overlaps the ZEB-binding ZV silencing element of Zp. Depending upon posttranslational modifications, Smad4 either competed with ZEB1 for binding or formed a complex with ZEB1 on the Zp ZV element in a cell-free assay system. In transiently transfected cells, exogenously expressed ZEB1 inhibited Smad-mediated transcriptional activation from Zp. We conclude that TGF-β induces EBV lytic reactivation via the canonical Smad pathway by activating BZLF1 gene expression through multiple SBEs acting in concert.

Cellular MicroRNAs 200b and 429 Regulate the Epstein-Barr Virus Switch between Latency and Lytic Replication

We previously showed that the cellular proteins ZEB1 and ZEB2/SIP1 both play key roles in regulating the latent-lytic switch of Epstein-Barr Virus (EBV) by repressing BZLF1 gene expression. We investigated here the effects of cellular microRNA (miRNA) 200 (miR200) family members on the EBV infection status of cells. We show that miR200b and miR429, but not miR200a, can induce EBV-positive cells into lytic replication by downregulating expression of ZEB1 and ZEB2, leading to production of infectious virus. The levels of miR200 family members in EBV-infected cells strongly negatively correlated with the levels of the ZEBs (e.g., -0.89 [P < 0.001] for miR429 versus ZEB1) and positively correlated with the degree of EBV lytic gene expression (e.g., 0.73 [P < 0.01] for miR429 versus BZLF1). The addition of either miR200b or miR429 to EBV-positive cells led to EBV lytic reactivation in a ZEB-dependent manner; inhibition of these miRNAs led to decreased EBV lytic gene expression. The degree of latent infection by an EBV mutant defective in the primary ZEB-binding site of the EBV BZLF1 promoter was not affected by the addition of these miRNAs. Furthermore, EBV infection of primary blood B cells led to downregulation of these miRNAs and upregulation of ZEB levels. Thus, we conclude that miRNAs 200b and 429 are key regulators via their effects on expression of ZEB1 and ZEB2 of the switch between latent and lytic infection by EBV and, therefore, potential targets for development of new lytic induction therapeutics with which to treat patients with EBV-associated malignancies.

Either ZEB1 or ZEB2/SIP1 Can Play a Central Role in Regulating the Epstein-Barr Virus Latent-Lytic Switch in a Cell-Type-Specific Manner

We previously reported that the cellular protein ZEB1 can repress expression of the Epstein-Barr virus (EBV) BZLF1 gene in transient transfection assays by directly binding its promoter, Zp. We also reported that EBV containing a 2-bp substitution mutation in the ZEB-binding ZV element of Zp spontaneously reactivated out of latency into lytic replication at a higher frequency than did wild-type EBV. Here, using small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies, we definitively show that ZEB1 is, indeed, a key player in maintaining EBV latency in some epithelial and B-lymphocytic cell lines. However, in other EBV-positive epithelial and B-cell lines, another zinc finger E-box-binding protein, ZEB2/SIP1, is the key player. Both ZEB1 and ZEB2 can bind Zp via the ZV element. In EBV-positive cells containing only ZEB1, knockdown of ZEB1 led to viral reactivation out of latency, with synthesis of EBV immediate-early and early lytic gene products. However, in EBV-positive cells containing both ZEBs, ZEB2, not ZEB1, was the primary ZEB family member bound to Zp. Knockdown of ZEB2, but not ZEB1, led to EBV lytic reactivation. Thus, we conclude that either ZEB1 or ZEB2 can play a central role in the maintenance of EBV latency, doing so in a cell-type-dependent manner.

Transactivators Zta and Rta of Epstein–Barr virus promote G0/G1 to S transition in Raji cells: A novel relationship between lytic virus and cell cycle

In the present study, we show that the treatment of Epstein–Barr virus (EBV) latently infected Raji cells with TPA/SB caused the cell growth arrest. The Zta-positive cells were predominantly enriched in G0/G1 phase of cell cycle. When Zta expression reached a maximal level, a fraction of Zta expressing cell population reentered S phase. Analysis of the expression pattern of a key set of cell cycle regulators revealed that the expression of Zta and Rta substantially interfered with the cell cycle regulatory machinery in Raji cells, strongly inhibiting the expression of Rb and p53 and inducing the expression of E2F1. Down-regulation of Rb was further demonstrated to be mediated by proteasomal degradation, and p53 and p21 affected at transcription level. The data indicate that both Zta and Rta promote entry into S phase of Raji cells. The important roles of Zta and Rta in EBV lytic reactivation were also demonstrated. Our finding suggests that these two transcriptional activators may act synergistically to govern the expression of downstream early and late genes as well as cellular genes and initiation of lytic cycle and manipulation of cell cycle regulatory mechanisms require the joint and interactive contributions of Rta and Zta.