Lytic and Latent Genetic Diversity of the Epstein-Barr Virus Reveals Raji-Related Variants from Southeastern Brazil Associated with Recombination Markers.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus etiologically associated with benign and malignant diseases. Since the pathogenic mechanisms of EBV are not fully understood, understanding EBV genetic diversity is an ongoing goal. Therefore, the present work describes the genetic diversity of the lytic gene BZLF1 in a sampling of 70 EBV-positive cases from southeastern Brazil. Additionally, together with the genetic regions previously characterized, the aim of the present study was to determine the impact of viral genetic factors that may influence EBV genetic diversity. Accordingly, the phylogenetic analysis of the BZLF1 indicated two main clades with high support, BZ-A and BZ-B (PP > 0.85). Thus, the BZ-A clade was the most diverse clade associated with the main polymorphisms investigated, including the haplotype Type 1 + V3 (p < 0.001). Furthermore, the multigene phylogenetic analysis (MLA) between BZLF1 and the oncogene LMP1 showed specific clusters, revealing haplotypic segregation that previous single-gene phylogenies from both genes failed to demonstrate. Surprisingly, the LMP1 Raji-related variant clusters were shown to be more diverse, associated with BZ-A/B and the Type 2/1 + V3 haplotypes. Finally, due to the high haplotypic diversity of the Raji-related variants, the number of DNA recombination-inducing motifs (DRIMs) was evaluated within the different clusters defined by the MLA. Similarly, the haplotype BZ-A + Raji was shown to harbor a greater number of DRIMs (p < 0.001). These results call attention to the high haplotype diversity of EBV in southeast Brazil and strengthen the hypothesis of the recombinant potential of South American Raji-related variants via the LMP1 oncogene.

Ceramide promotes lytic reactivation of Epstein-Barr virus in gastric carcinoma.

Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.

Construction of rBCG carrying the IL-2-BZLF1 fusion gene and its immunological function.

In this research, a recombinant Bacillus Calmette Guerin (rBCG) vector vaccine carrying a human IL-2 and EBV BZLF1 fusion gene (IL-2-BZLF1-rBCG) was constructed. The IL-2-BZLF1-rBCG construct was successfully generated and stably expressed the IL-2 and BZLF1 proteins. IL-2-BZLF1-rBCG activated the immune system and promoted the secretion of IFN-γ and TNF-α by CD4+ and CD8+ T cells. IL-2-BZLF1-rBCG activated lymphocytes to effectively kill EBV-positive NPC cells in vitro. Additionally, IL-2-BZLF1-rBCG stimulated the proliferation of NK cells and lymphocytes in vivo, activated related immune responses, and effectively treated EBV-positive NPC. The immune response to and pharmacological effect of IL-2-BZLF1-rBCG were explored in vitro and in vivo to provide a theoretical and experimental basis for the prevention and treatment of EBV-positive tumors with an rBCG vector vaccine. KEY POINTS: • rBCG with human IL-2 and BZLF1 of EB virus was constructed • The IL-2-BZLF1 fusion gene was stably expressed with rBCG • rBCG with IL-2-BZLF1 has an obvious immune response in vitro and in vivo.

A library-derived peptide inhibitor of the BZLF1 transcription factor.

Transcription factor dysregulation is associated with many diseases, including cancer. Peptide-based molecules are increasingly recognised as important modulators of difficult intracellular protein-protein interaction targets, with peptide library screening consequently proven to be a viable strategy in developing inhibitors against a wide range of transcription factors (TFs). However, current strategies simply select the highest affinity of binding to a target TF rather than the ability to inhibit TF function. Here, we utilise our Transcription Block Survival (TBS) screening platform to enable high-throughput identification of peptides that inhibit TFs from binding to cognate DNA sites, hence inhibiting functionality. In this study, we explore whether the TBS can be expanded to derive a potent and functional peptide inhibitor of the BZLF1 transcription factor. The library-derived peptide, AcidicW, is shown to form a more stable dimer with BZLF1 than the BZLF1 homodimer, with a thermal denaturation temperature exceeding 80°C. AcidicW can also functionally inhibit the BZLF1:TRE DNA interaction with high potency and an IC50 of 612 nM.

An in-silico insight into the predictive interaction of Apolipoprotein-E with Epstein-Barr virus proteins and their probable role in mediating Alzheimer's disease.

Recent reports suggest that persistent Epstein-Barr virus (EBV) infection and its recurrent reactivation could instigate the formation of proteinaceous plaques in the brain: a hallmark of Alzheimer's disease (AD). Interestingly, a major genetic risk factor of AD, the apolipoprotein E (ApoE), could also influence the outcome of EBV infection in an individual. The ApoE is believed to influence the proteinaceous plaque clearance from the brain, and its defective functioning could result in the aggregate deposition. The persistent presence of EBV infection in a genetically predisposed individual could create a perfect recipe for severe neurodegenerative consequences. Therefore, in the present study, we investigated the possible interactions between ApoE and various EBV proteins using computational tools. Our results showed possibly stable de-novo interactions between the C-terminal domain of ApoE3 and EBV proteins: EBV nuclear antigen-1 (EBNA1) and BamHI Z fragment leftward open reading frame-1 (BZLF1). The EBNA1 protein of EBV plays a crucial role in establishing latency and replication of the virus. Whereas BZLF1 is involved in the lytic replication cycle. The proposed interaction of EBV proteins at the ligand-binding site of ApoE3 on CTD could interfere with- its capability to sequester amyloid fragments and, hence their clearance from the brain giving rise to AD pathology. This study provides a new outlook on EBV's underexplored role in AD development and paves the way for novel avenues of investigation which could further our understanding of AD pathogenesis.Communicated by Ramaswamy H. Sarma[Figure: see text].

Recent Advances in Assessing the Clinical Implications of Epstein-Barr Virus Infection and Their Application to the Diagnosis and Treatment of Nasopharyngeal Carcinoma.

Reports about the oncogenic mechanisms underlying nasopharyngeal carcinoma (NPC) have been accumulating since the discovery of Epstein-Barr virus (EBV) in NPC cells. EBV is the primary causative agent of NPC. EBV-host and tumor-immune system interactions underlie the unique representative pathology of NPC, which is an undifferentiated cancer cell with extensive lymphocyte infiltration. Recent advances in the understanding of immune evasion and checkpoints have changed the treatment of NPC in clinical settings. The main EBV genes involved in NPC are LMP1, which is the primary EBV oncogene, and BZLF1, which induces the lytic phase of EBV. These two multifunctional genes affect host cell behavior, including the tumor-immune microenvironment and EBV behavior. Latent infections, elevated concentrations of the anti-EBV antibody and plasma EBV DNA have been used as biomarkers of EBV-associated NPC. The massive infiltration of lymphocytes in the stroma suggests the immunogenic characteristics of NPC as a virus-infected tumor and, at the same time, also indicates the presence of a sophisticated immunosuppressive system within NPC tumors. In fact, immune checkpoint inhibitors have shown promise in improving the prognosis of NPC patients with recurrent and metastatic disease. However, patients with advanced NPC still require invasive treatments. Therefore, there is a pressing need to develop an effective screening system for early-stage detection of NPC in patients. Various modalities, such as nasopharyngeal cytology, cell-free DNA methylation, and deep learning-assisted nasopharyngeal endoscopy for screening and diagnosis, have been introduced. Each modality has its advantages and disadvantages. A reciprocal combination of these modalities will improve screening and early diagnosis of NPC.

High Predictive Value of the Soluble ZEBRA Antigen (Epstein-Barr Virus Trans-Activator Zta) in Transplant Patients with PTLD.

The ZEBRA (Z EBV replication activator) protein is the major transcription factor of EBV, expressed upon EBV lytic cycle activation. An increasing body of studies have highlighted the critical role of EBV lytic infection as a risk factor for lymphoproliferative disorders, such as post-transplant lymphoproliferative disease (PTLD). We studied 108 transplanted patients (17 PTLD and 91 controls), retrospectively selected from different hospitals in France and in the Netherlands. The majority of PTLD were EBV-positive diffuse large B-cell lymphomas, five patients experienced atypical PTLD forms (EBV-negative lymphomas, Hodgkin's lymphomas, and T-cell lymphomas). Fourteen patients among the seventeen who developed a pathologically confirmed PTLD were sZEBRA positive (soluble ZEBRA, plasma level above 20 ng/mL, measured by an ELISA test). The specificity and positive predictive value (PPV) of the sZEBRA detection in plasma were 98% and 85%, respectively. Considering a positivity threshold of 20 ng/mL, the sensitivity of the sZEBRA was 82.35% and the specificity was 94.51%. The mean of the sZEBRA values in the PTLD cases were significantly higher than in the controls (p < 0.0001). The relevance of the lytic cycle and, particularly, the role of ZEBRA in lymphomagenesis is a new paradigm pertaining to the prevention and treatment strategies for PTLD. Given the high-specificity and the predictive values of this test, it now appears relevant to investigate the lytic EBV infection in transplanted patients as a prognostic biomarker.

Andrographolide Inhibits Epstein-Barr Virus Lytic Reactivation in EBV-Positive Cancer Cell Lines through the Modulation of Epigenetic-Related Proteins.

Reactivation of Epstein-Barr virus (EBV) is associated with EBV-associated malignancies and is considered to be a benefit target for treatment. Andrographolide is claimed to have antiviral and anti-tumor activities. Therefore, this study aimed to investigate the effect of andrographolide on the inhibition of EBV lytic reactivation in EBV-positive cancer cells. The cytotoxicity of andrographolide was firstly evaluated in EBV-positive cancer cells; P3HR1, AGS-EBV and HONE1-EBV cells, using an MTT assay. Herein, the spontaneous expression of EBV lytic genes; BALF5, BRLF1 and BZLF1, was significantly inhibited in andrographolide-treated cells. Accordingly, andrographolide inhibited the expression of Zta and viral production in sodium butyrate (NaB)-induced EBV lytic reactivation. Additionally, proteomics and bioinformatics analysis revealed the differentially expressed proteins that inhibit EBV lytic reactivation in all treated cell lines were functionally related with the histone modifications and chromatin organization, such as histone H3-K9 modification and histone H3-K27 methylation. Taken together, andrographolide inhibits EBV reactivation in EBV-positive cancer cells by inhibiting EBV lytic genes, probably, through the histone modifications.

Estrogen induces the expression of EBV lytic protein ZEBRA, a marker of poor prognosis in nasopharyngeal carcinoma.

Several epidemiological studies have suggested that Epstein-Barr virus (EBV) lytic infection is essential for the development of nasopharyngeal carcinoma (NPC), as the elevation of antibody titers against EBV lytic proteins is a common feature of NPC. Although ZEBRA protein is a key trigger for the initiation of lytic infection, whether its expression affects the prognosis and pathogenesis of NPC remains unclear. In this study, 64 NPC biopsy specimens were analyzed using immunohistochemistry. We found that ZEBRA was significantly associated with a worsening of progression-free survival in NPC (adjusted hazard ratio, 3.58; 95% confidence interval, 1.08-11.87; p = 0.037). Moreover, ZEBRA expression positively correlated with key endocrinological proteins, estrogen receptor α, and aromatase. The transcriptional level of ZEBRA is activated by estrogen in an estrogen receptor α-dependent manner, resulting in an increase in structural gene expression levels and extracellular virus DNA copy number in NPC cell lines, reminiscent of lytic infection. Interestingly, it did not suppress cellular proliferation or increase apoptosis, in contrast with cells treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, indicating that viral production induced by estrogen is not a cell lytic phenomenon. Our results suggest that intratumoral estrogen overproduced by aromatase could induce ZEBRA expression and EBV reactivation, contributing to the progression of NPC.

Targeted Delivery of BZLF1 to DEC205 Drives EBV-Protective Immunity in a Spontaneous Model of EBV-Driven Lymphoproliferative Disease.

Epstein-Barr virus (EBV) is a human herpes virus that infects over 90% of the world's population and is linked to development of cancer. In immune-competent individuals, EBV infection is mitigated by a highly efficient virus-specific memory T-cell response. Risk of EBV-driven cancers increases with immune suppression (IS). EBV-seronegative recipients of solid organ transplants are at high risk of developing post-transplant lymphoproliferative disease (PTLD) due to iatrogenic IS. While reducing the level of IS may improve EBV-specific immunity and regression of PTLD, patients are at high risk for allograft rejection and need for immune-chemotherapy. Strategies to prevent PTLD in this vulnerable patient population represents an unmet need. We have previously shown that BZLF1-specific cytotoxic T-cell (CTL) expansion following reduced IS correlated with immune-mediated PTLD regression and improved patient survival. We have developed a vaccine to bolster EBV-specific immunity to the BZLF1 protein and show that co-culture of dendritic cells (DCs) loaded with a αDEC205-BZLF1 fusion protein with peripheral blood mononuclear cells (PMBCs) leads to expansion and increased cytotoxic activity of central-effector memory CTLs against EBV-transformed B-cells. Human-murine chimeric Hu-PBL-SCID mice were vaccinated with DCs loaded with αDEC205-BZLF1 or control to assess prevention of fatal human EBV lymphoproliferative disease. Despite a profoundly immunosuppressive environment, vaccination with αDEC205-BZLF1 stimulated clonal expansion of antigen-specific T-cells that produced abundant IFNγ and significantly prolonged survival. These results support preclinical and clinical development of vaccine approaches using BZLF1 as an immunogen to harness adaptive cellular responses and prevent PTLD in vulnerable patient populations.

Identification of an N6-methyladenosine-mediated positive feedback loop that promotes Epstein-Barr virus infection.

N6-methyladenosine (m6A) is among the most abundant mRNA modifications, particularly in eukaryotes, and is found in mammals, plants, and even some viruses. Although essential for the regulation of many biological processes, the exact role of m6A modification in virus-host interaction remains largely unknown. Here, using m6A -immunoprecipitation and sequencing, we find that Epstein-Barr virus (EBV) infection decreases the m6A modification of transcriptional factor KLF4 mRNA and subsequently increases its protein level. Mechanistically, EBV immediate-early protein BZLF1 interacts with the promoter of m6A methyltransferase METTL3, inhibiting its expression. Subsequently, the decrease of METTL3 reduces the level of KLF4 mRNA m6A modification, preventing its decay by the m6A reader protein YTHDF2. As a result, KLF4 protein level is upregulated and, in turn, promotes EBV infection of nasopharyngeal epithelial cells. Thus, our results suggest the existence of a positive feedback loop formed between EBV and host molecules via cellular mRNA m6A levels, and this feedback loop acts to facilitate viral infection. This mechanism contains multiple potential targets for controlling viral infectious diseases.

CXCR4 induces cell autophagy and maintains EBV latent infection in EBVaGC.

Rationale: Epstein-Barr virus (EBV) is found in ~7% of gastric carcinoma cases worldwide, and all tumour cells harbour the clonal EBV genome. EBV can regulate pathways and protein expression to induce gastric carcinoma; however, the molecular mechanism underlying EBV-associated gastric carcinoma (EBVaGC) remains elusive. Methods: GEO microarray and molecular experiments were performed to compare CXCR4 expression between EBV-positive and EBV-negative gastric carcinoma (EBVnGC). Transfections with LMP2A plasmid or siRNA were carried out to assess the role of LMP2A in CXCR4 expression. The effects and mechanisms of CXCR4 on cell autophagy were analysed in vitro using molecular biological and cellular approaches. Additionally, we also determined the regulatory role of CXCR4 in latent EBV infection. Results: CXCR4 expression was significantly upregulated in EBVaGC tissues and cell lines. LMP2A could induce AKT phosphorylation to increase NRF1 expression, thereby binding to the CXCR4 promoter to increase its transcriptional level. Moreover, CXCR4 promoted ZEB1 expression to upregulate ATG7 synthesis, which could then activate autophagy. Moreover, CXCR4 increased the number of cells entering the G2/M phase and inhibited cell apoptosis via the autophagy pathway. Finally, CXCR4 knockdown was associated with elevated BZLF1 expression, but this effect was not influenced by autophagy. Conclusions: Our data suggested new roles for CXCR4 in autophagy and EBV replication in EBVaGC, which further promoted cell survival and persistent latent infection. These new findings can lead to further CXCR4-based anticancer therapy.

Reactivation of Epstein-Barr Virus by HIF-1α Requires p53.

We previously reported that the cellular transcription factor hypoxia-inducible factor 1α (HIF-1α) binds a hypoxia response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. In this study, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1α-stabilizing drugs: the iron chelator deferoxamine (Desferal [DFO]), a neddylation inhibitor (pevonedistat [MLN-4924]), and a prolyl hydroxylase inhibitor (roxadustat [FG-4592]). DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1α and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with short hairpin RNAs (shRNAs). Likewise, HIF-1α failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1α to Zp in chromatin immunoprecipitation (ChIP) assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated, thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1α requires phosphorylated, wild-type p53 as a coactivator, with HIF-1α binding recruiting p53 to Zp.IMPORTANCE EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's BZLF1 gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1α binds the BZLF1 gene promoter, inducing Zta synthesis, and HIF-1α-stabilizing drugs can induce EBV reactivation. In this study, we determined which EBV-positive cell lines are reactivated by classes of HIF-1α-stabilizing drugs. We found, unexpectedly, that HIF-1α-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1α-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.

Oncogenic Properties of the EBV ZEBRA Protein.

Epstein Barr Virus (EBV) is one of the most common human herpesviruses. After primary infection, it can persist in the host throughout their lifetime in a latent form, from which it can reactivate following specific stimuli. EBV reactivation is triggered by transcriptional transactivator proteins ZEBRA (also known as Z, EB-1, Zta or BZLF1) and RTA (also known as BRLF1). Here we discuss the structural and functional features of ZEBRA, its role in oncogenesis and its possible implication as a prognostic or diagnostic marker. Modulation of host gene expression by ZEBRA can deregulate the immune surveillance, allow the immune escape, and favor tumor progression. It also interacts with host proteins, thereby modifying their functions. ZEBRA is released into the bloodstream by infected cells and can potentially penetrate any cell through its cell-penetrating domain; therefore, it can also change the fate of non-infected cells. The features of ZEBRA described in this review outline its importance in EBV-related malignancies.

Targeting Epstein-Barr Virus in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection in regions in which it is endemic, including Southern China and Southeast Asia. The high mortality rates of NPC patients with advanced and recurrent disease highlight the urgent need for effective treatments. While recent genomic studies have revealed few druggable targets, the unique interaction between the EBV infection and host cells in NPC strongly implies that targeting EBV may be an efficient approach to cure this virus-associated cancer. Key features of EBV-associated NPC are the persistence of an episomal EBV genome and the requirement for multiple viral latent gene products to enable malignant transformation. Many translational studies have been conducted to exploit these unique features to develop pharmaceutical agents and therapeutic strategies that target EBV latent proteins and induce lytic reactivation in NPC. In particular, inhibitors of the EBV latent protein EBNA1 have been intensively explored, because of this protein's essential roles in maintaining EBV latency and viral genome replication in NPC cells. In addition, recent advances in chemical bioengineering are driving the development of therapeutic agents targeting the critical functional regions of EBNA1. Promising therapeutic effects of the resulting EBNA1-specific inhibitors have been shown in EBV-positive NPC tumors. The efficacy of multiple classes of EBV lytic inducers for NPC cytolytic therapy has also been long investigated. However, the lytic-induction efficiency of these compounds varies among different EBV-positive NPC models in a cell-context-dependent manner. In each tumor, NPC cells can evolve and acquire somatic changes to maintain EBV latency during cancer progression. Unfortunately, the poor understanding of the cellular mechanisms regulating EBV latency-to-lytic switching in NPC cells limits the clinical application of EBV cytolytic treatment. In this review, we discuss the potential approaches for improvement of the above-mentioned EBV-targeting strategies.

BZLF1 transcript variants in Epstein-Barr virus-positive epithelial cell lines.

Epstein-Barr virus (EBV) is a widely prevalent pathogen currently infecting over 90% of the human population and is associated with various lymphomas and carcinomas. Lytic replication of EBV is regulated by the expression of the immediate-early genes BZLF1 and BRLF1. In B lymphocytes, BZLF1 transcripts have been shown to be processed to a fully spliced form, as well as zDelta, a spliced variant containing only the first and third exons. While splice variants have been reported in nasopharyngeal carcinoma biopsies, alternative splicing of BZLF1 in EBV-positive epithelial cell lines has not yet been characterized. In this study, we identified the consistent expression of three distinct BZLF1 transcripts in the EBV-positive epithelial cell lines D98/HR1, AGS-BDneo, and AGS-BX1. These BZLF1 transcripts consisted of not only the normally spliced variant but also a completely unspliced and a spliced variant containing exons one and three only. In contrast, we detected only the normally spliced version of the BZLF1 transcript in B-cell lines (B95-8, IM-9, Raji and Daudi). Previous work has also demonstrated that inhibition of the mTOR pathway, via rapamycin, altered total levels of BZLF1 transcripts. We examined the production of specific transcript variants under rapamycin treatment and found that rapamycin alters the production of transcripts in a cell-type, as well as transcripts in variant-type, manners. The expression of these transcript variants may play a role in modulating the replication cycle of EBV within epithelial cells.

Interaction of phospholipid scramblase 1 with the Epstein-Barr virus protein BZLF1 represses BZLF1-mediated lytic gene transcription.

Human phospholipid scramblase 1 (PLSCR1) is strongly expressed in response to interferon (IFN) treatment and viral infection, and PLSCR1 has been suggested to play an important role in IFN-dependent antiviral responses. In this study, we showed that the basal expression of PLSCR1 was significantly elevated in Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC). PLSCR1 was observed to directly interact with the EBV immediate-early transactivator BZLF1 in vitro and in vivo, and this interaction repressed the BZLF1-mediated transactivation of an EBV lytic BMRF1 promoter construct. In addition, PLSCR1 expression decreased the BZLF1-mediated up-regulation of lytic BMRF1 mRNA and protein expression in WT and PLSCR1-knockout EBV-infected NPC cells. Furthermore, we showed that PLSCR1 represses the interaction between BZLF1 and CREB-binding protein (CBP), which enhances the BZLF1-mediated transactivation of EBV lytic promoters. These results reveal for the first time that PLSCR1 specifically interacts with BZLF1 and negatively regulates its transcriptional regulatory activity by preventing the formation of the BZLF1-CBP complex. This interaction may contribute to the establishment of latent EBV infection in EBV-infected NPC cells.

Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation.

Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication. Lytic reactivation starts with derepression of the Zp promoter controlling BZLF1 gene expression, which binds and is activated by the c-Jun transcriptional activator. Here, we identified the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation. Silencing of ARKL1 in the context of EBV-positive gastric carcinoma (AGS) cells, nasopharyngeal carcinoma (NPC43) cells, and B (M81) cells led to increased lytic protein expression, whereas overexpression inhibited BZLF1 expression. Similar effects of ARKL1 modulation were seen on BZLF1 transcripts as well as on Zp activity in Zp reporter assays, showing that ARKL1 repressed Zp. Proteomic profiling of ARKL1-host interactions identified c-Jun as an ARKL1 interactor, and reporter assays for Jun transcriptional activity showed that ARKL1 inhibited Jun activity. The ARKL1-Jun interaction required ARKL1 sequences that we previously showed mediated binding to the CK2 kinase regulatory subunit CK2ß, suggesting that CK2ß might mediate the ARKL1-Jun interaction. This model was supported by the findings that silencing of CK2ß, but not the CK2α catalytic subunit, abrogated the ARKL1-Jun interaction and phenocopied ARKL1 silencing in promoting EBV reactivation. Additionally, ARKL1 was associated with Zp in reporter assays and this was increased by additional CK2ß. Together, the data indicate that ARKL1 is a negative regulator of Zp and EBV reactivation that acts by inhibiting Jun activity through a CK2ß-mediated interaction.IMPORTANCE Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication and is associated with several types of cancer. We have identified a cellular protein (ARKL1) that acts to repress the reactivation of EBV from the latent to the lytic cycle. We show that ARKL1 acts to repress transcription of the EBV lytic switch protein by inhibiting the activity of the cellular transcription factor c-Jun. This not only provides a new mechanism of regulating EBV reactivation but also identifies a novel cellular function of ARKL1 as an inhibitor of Jun-mediated transcription.

A Noncanonical Basic Motif of Epstein-Barr Virus ZEBRA Protein Facilitates Recognition of Methylated DNA, High-Affinity DNA Binding, and Lytic Activation.

The pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade.IMPORTANCE The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

Hydroa vacciniforme: a distinctive form of Epstein-Barr virus-associated T-cell lymphoproliferative disorders.

Hydroa vacciniforme (HV) is a cutaneous subset of Epstein-Barr virus (EBV)-associated T/NK lymphoproliferative disorders (LPDs). Our previous case series study clearly showed a clinical spectrum of EBV-associated T/NK LPDs including HV, hypersensitivity to mosquito bites (HMB), chronic active EBV infection (CAEBV), and hemophagocytic lymphohistiocytosis (HLH). Patients with HV are divided into two groups: a benign subtype designated "classic HV" (cHV) and more serious systemic HV (sHV), also called "HV-like LPD" in the 2017 World Health Organization (WHO) classification. Patients with cHV usually have an increased number of EBV-infected γδT cells and patients with sHV without HMB are further classified into two groups: γδT-cell- and αßT-cell-dominant types. Patients with HMB, with or without HV-like eruptions, have an increased number of EBV-infected NK cells in the blood. Patients with cHV and γδT-cell-dominant sHV show a favourable prognosis, but the other subtypes such as αßT-cell-dominant sHV and HMB have a poor prognosis with mortality rates of 11.5 and 3.51 per 100 person-years, respectively. In addition to the clinical subtypes and the dominant lymphocyte subsets, the poor prognostic indicators include onset age over nine years and expression of the reactivation marker, BZLF1 mRNA. No prognostic correlation has been reported for anti-EBV antibody titres or EBV DNA load. The clinical subtypes and their prognostic factors should be considered for therapeutic interventions.