Lytic Epstein-Barr Virus Infection Research Articles

Suppression of IFN-β production by Epstein-Barr virus lytic transactivator Zta

Abstract Interferon β (IFN-β) production is an innate antiviral immune response against virus infection and lytic virus production. Several molecular mechanisms, including the sensing of cytosolic double-stranded DNA (dsDNA) by cyclic GMP-AMP synthase (cGAS), have been described to induce IFN-β production. Epstein-Barr virus (EBV) is a γ-herpesvirus that infects most of the population in the world and is associated with the development of various lymphoid and epithelial malignancies in a subset of people. In this study, we identified and characterized EBV lytic transactivator Zta as a potent suppressor of IFN-β production. EBV lytic infection was shown to suppress IFN-β production. To identify the viral proteins that mediate this suppression, a functional screen was performed using a dual-luciferase reporter system and an expression library of all EBV proteins. In this screen EBV lytic transactivator Zta was found to be capable of potently suppressing IFN-β production induced by different stimuli of interferon regulatory factor 3 (IRF3) activation. Zta is previously known to directly interact with and suppress IRF7, but its impact on IRF3 and the mode of action have not been demonstrated. We showed that Zta prevented the activation of IRF3 and IFN-β promoter by cGAS and STING (stimulator of interferon genes). Mechanistically, Zta suppressed the phosphorylation of IRF3 and its upstream interacting partner TANK-binding kinase 1 (TBK1), leading to the inhibition of IRF3 activity and nuclear translocation. Therefore, Zta may help EBV to evade host immunity during lytic infection by circumventing IRF3 activation and thus IFN-β production. Our findings revealed a new mechanism for EBV evasion of innate immunity.

Epstein-Barr virus lytic infection promotes activation of Toll-like receptor 8 innate immune response in systemic sclerosis monocytes

BackgroundMonocytes/macrophages are activated in several autoimmune diseases, including systemic sclerosis (scleroderma; SSc), with increased expression of interferon (IFN)-regulatory genes and inflammatory cytokines, suggesting dysregulation of the innate immune response in autoimmunity. In this study, we investigated whether the lytic form of Epstein-Barr virus (EBV) infection (infectious EBV) is present in scleroderma monocytes and contributes to their activation in SSc.MethodsMonocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of the CD19+ cell fraction, using CD14/CD16 negative-depletion. Circulating monocytes from SSc and healthy donors (HDs) were infected with EBV. Gene expression of innate immune mediators were evaluated in EBV-infected monocytes from SSc and HDs. Involvement of Toll-like receptor (TLR)8 in viral-mediated TLR8 response was investigated by comparing the TLR8 expression induced by infectious EBV to the expression stimulated by CL075/TLR8/agonist-ligand in the presence of TLR8 inhibitor in THP-1 cells.ResultsInfectious EBV strongly induced TLR8 expression in infected SSc and HD monocytes in vitro. Markers of activated monocytes, such as IFN-regulated genes and chemokines, were upregulated in SSc- and HD-EBV-infected monocytes. Inhibiting TLR8 expression reduced virally induced TLR8 in THP-1 infected cells, demonstrating that innate immune activation by infectious EBV is partially dependent on TLR8. Viral mRNA and proteins were detected in freshly isolated SSc monocytes. Microarray analysis substantiated the evidence of an increased IFN signature and altered level of TLR8 expression in SSc monocytes carrying infectious EBV compared to HD monocytes.ConclusionThis study provides the first evidence of infectious EBV in monocytes from patients with SSc and links EBV to the activation of TLR8 and IFN innate immune response in freshly isolated SSc monocytes. This study provides the first evidence of EBV replication activating the TLR8 molecular pathway in primary monocytes. Immunogenicity of infectious EBV suggests a novel mechanism mediating monocyte inflammation in SSc, by which EBV triggers the innate immune response in infected cells.

EBV Infection and Glucose Metabolism in Nasopharyngeal Carcinoma.

To establish persistent infection in cells, viruses evolve strategies to alter host cellular pathways to regulate cell proliferation and energy metabolism which support viral infection. Epstein-Barr virus (EBV) undergoes both lytic and latent infection to achieve persistent and lifelong infection in human. EBV readily infects human B cells, driving their transformation to proliferative lymphoblastoid cell lines (LCL), and eventually establishes lifelong latent infection in memory B cells. In contrary, EBV undergoes lytic replication upon infection into normal epithelial cells which is essential for the replication of EBV genome and production of infectious viral particles for transmission through saliva. EBV shuttles between B cells and epithelial cells to complete its infection cycle. EBV infection is closely associated with nasopharyngeal carcinoma (NPC) and is present in practically 100% of undifferentiated NPC. In contrast to undergo lyticinfection of normal pharyngeal epithelium, EBV establishes latent infection in NPC. The switch from lytic infection to latent infection may represent an early and essential step in the development of NPC. Recent studies in both B cells and NPC cells latently infected with EBV reveal alterations in cell metabolism to support persistentand latent EBV infection. Events underlying the switching of lytic to latent EBV infection in NPC cells are largely undefined. Molecular events and alterations of cell metabolism are likely to play crucial roles in switching EBV infection from lytic to latent in NPC cells. Latent EBV infection and expression of viral genes, including LMP1, LMP2, and possibly EBV-encoded micro RNAs, may play essential roles in alterations of cell metabolism to support NPC pathogenesis. Alteration of energy metabolism is an essential hallmark of cancer. The role of altered energy metabolism in host cells in modulating latent and lytic EBV infection in NPC cells is unclear. In this review, we will discuss the impact of genetic alterations in NPC to module cellular metabolism and its influence on latent infection and lytic reactivation of EBV infection in NPC cells. In particular, the role of EBV-encoded genes in driving glucose metabolism and their contribution to NPC pathogenesis will be discussed. This new perspective on the interplay between EBV infection and altered host metabolic pathways in NPC pathogenesis may offer novel and effective therapeutic strategies in the treatment of NPC and other EBV-associated malignancies.

Quantitative multi-target RNA profiling in Epstein-Barr virus infected tumor cells

Epstein-Barr virus (EBV) is etiologically linked to multiple acute, chronic and malignant diseases. Detection of EBV-RNA transcripts in tissues or biofluids besides EBV-DNA can help in diagnosing EBV related syndromes. Sensitive EBV transcription profiling yields new insights on its pathogenic role and may be useful for monitoring virus targeted therapy. Here we describe a multi-gene quantitative RT-PCR profiling method that simultaneously detects a broad spectrum (n=16) of crucial latent and lytic EBV transcripts. These transcripts include (but are not restricted to), EBNA1, EBNA2, LMP1, LMP2, BARTs, EBER1, BARF1 and ZEBRA, Rta, BGLF4 (PK), BXLF1 (TK) and BFRF3 (VCAp18) all of which have been implicated in EBV-driven oncogenesis and viral replication. With this method we determine the amount of RNA copies per infected (tumor) cell in bulk populations of various origin. While we confirm the expected RNA profiles within classic EBV latency programs, this sensitive quantitative approach revealed the presence of rare cells undergoing lytic replication. Inducing lytic replication in EBV tumor cells supports apoptosis and is considered as therapeutic approach to treat EBV-driven malignancies. This sensitive multi-primed quantitative RT-PCR approach can provide broader understanding of transcriptional activity in latent and lytic EBV infection and is suitable for monitoring virus-specific therapy responses in patients with EBV associated cancers.

BZLF1 Attenuates Transmission of Inflammatory Paracrine Senescence in Epstein-Barr Virus-Infected Cells by Downregulating Tumor Necrosis Factor Alpha

Recent studies have shown that inflammatory responses trigger and transmit senescence to neighboring cells and activate the senescence-associated secretory phenotype (SASP). Latent Epstein-Barr virus (EBV) infection induces increased secretion of several inflammatory factors, whereas lytic infections evade the antiviral inflammatory response. However, the changes in and roles of the inflammatory microenvironment during the switch between EBV life cycles remain unknown. In the present study, we demonstrate that latent EBV infection in EBV-positive cells triggers the SASP in neighboring epithelial cells. In contrast, lytic EBV infection abolishes this phenotype. BZLF1 attenuates the transmission of paracrine senescence during lytic EBV infection by downregulating tumor necrosis factor alpha (TNF-α) secretion. A mutant BZLF1 protein, BZLF1Δ207-210, that cannot inhibit TNF-α secretion while maintaining viral transcription, fails to block paracrine senescence, whereas a neutralizing antibody against TNF-α is sufficient to restore its inhibition. Furthermore, latent EBV infection induces oxidative stress in neighboring cells, while BZLF1-mediated downregulation of TNF-α reduces reactive oxygen species (ROS) levels in neighboring cells, and ROS scavengers alleviate paracrine senescence. These results suggest that lytic EBV infection attenuates the transmission of inflammatory paracrine senescence through BZLF1 downregulation of TNF-α secretion and alters the inflammatory microenvironment to allow virus propagation and persistence. The senescence-associated secretory phenotype (SASP), an important tumorigenic process, is triggered and transmitted by inflammatory factors. The different life cycles of Epstein-Barr virus (EBV) infection in EBV-positive cells employ distinct strategies to modulate the inflammatory response and senescence. The elevation of inflammatory factors during latent EBV infection promotes the SASP in uninfected cells. In contrast, during the viral lytic cycle, BZLF1 suppresses the production of TNF-α, resulting in the attenuation of paracrine inflammatory senescence. This finding indicates that EBV evades inflammatory senescence during lytic infection and switches from facilitating tumor-promoting SASP to generating a virus-propagating microenvironment, thereby facilitating viral spread in EBV-associated diseases.

Association of GATA2 Deficiency With Severe Primary Epstein-Barr Virus (EBV) Infection and EBV-associated Cancers.

Most patients infected with Epstein-Barr virus (EBV) are asymptomatic, have nonspecific symptoms, or have self-limiting infectious mononucleosis. EBV, however, may result in severe primary disease or cancer. We report EBV diseases associated with GATA2 deficiency at one institution and describe the hematology, virology, and cytokine findings. Seven patients with GATA2 deficiency developed severe EBV disease. Three presented with EBV infectious mononucleosis requiring hospitalization, 1 had chronic active EBV disease (B-cell type), 1 had EBV-associated hydroa vacciniforme-like lymphoma with hemophagocytic lymphohistiocytosis, and 2 had EBV-positive smooth muscle tumors. Four of the 7 patients had severe warts and 3 had disseminated nontuberculous mycobacterial infections. All of the patients had low numbers of monocytes, B cells, CD4 T cells, and natural killer cells. All had elevated levels of EBV in the blood; 2 of 3 patients tested had expression of the EBV major immediate-early gene in the blood indicative of active EBV lytic infection. Mean plasma levels of tumor necrosis factor α, interferon γ, and interferon gamma-induced protein 10 were higher in patients with GATA2 deficiency than in controls. GATA2 is the first gene associated with EBV hydroa vacciniforme-like lymphoma. GATA2 deficiency should be considered in patients with severe primary EBV infection or EBV-associated cancer, especially in those with disseminated nontuberculous mycobacterial disease and warts.

Cordycepin enhances Epstein–Barr virus lytic infection and Epstein–Barr virus-positive tumor treatment efficacy by doxorubicin

The consistent latent presence of Epstein–Barr virus (EBV) in tumor cells offers potential for virus-targeted therapies. The switch from the latent form of EBV to the lytic form in tumor cells can lead to tumor cell lysis. In this study, we report that a natural small molecule compound, cordycepin, can induce lytic EBV infection in tumor cells. Subsequently, we demonstrate that cordycepin can enhance EBV reactivating capacity and EBV-positive tumor cell killing ability of low dose doxorubicin. The combination of cordycepin and doxorubicin phosphorylates CCAAT/enhancer binding protein β (C/EBPβ) through protein kinase C (PKC)-p38 mitogen activated protein kinases (p38 MAPK) signaling pathway, and C/EBPβ is required for the activation of lytic EBV infection. Most importantly, an in vivo experiment demonstrates that the combination of cordycepin and doxorubicin is more effective in inhibiting tumor growth in SCID mice than is doxorubicin alone. Our findings establish that cordycepin can enhance the efficacy of conventional chemotherapy for treatment of EBV-positive tumors.

Agents and Approaches for Lytic Induction Therapy of Epstein-Barr Virus Associated Malignancies

Epstein-Barr virus (EBV) is causally associated with multiple cancers of epithelial and lymphoid origin. Generally, EBV establishes a lifelong quiescent homeostasis (latency) exerting its immune modulating and oncogenic properties only under certain conditions. Lytic replicative EBV infection is restricted to differentiated oropharyngeal epithelia and plasma B-cells. In EBV-associated tumor cells viral gene expression is limited to defined latency programs, without overt virus replication. Artificial triggering of EBV lytic replication with chemical agents (drugs) forms the basis of cytolytic virus activation (CLVA) therapy currently under clinical investigation for treating EBV-positive malignancies. Reactivation from latency requires expression of viral transactivator proteins BZLF1 (Zta) and BRLF1 (Rta). In tumor cells, the BZLF1 promoter (Zp) is highly methylated and generally inactive. However, Zp can be triggered into a self-enhancing activation loop in response to chemical or biological agents, including cytostatic drugs and epigenetic modifiers. This process is orchestrated by Zp-binding cellular transcription factors, such as myocyte enhancer factor 2 (MEF2), specificity protein 1 (SP1), and zinc finger E-box binding homeobox (ZEB) proteins. Understanding the mechanism of lytic induction by chemical inducers is essential for developing effective therapies. This review provide a short overview of EBV biology and will focus on branded and novel agents (drugs) used for EBV lytic induction and discuss molecular mechanisms by which the EBV lytic switch is triggered and controlled.

Impaired Cytokine Responses to Epstein-Barr Virus Antigens in Systemic Lupus Erythematosus Patients.

We analyzed cytokine responses against latent and lytic Epstein-Barr virus (EBV) antigens in systemic lupus erythematosus (SLE) patients and healthy controls (HCs) to obtain an overview of the distinctive immune regulatory response in SLE patients and to expand the previously determined impaired EBV-directed T-cell response. The concentrations of 14 cytokines (IL2, IL4, IL5, IL6, IL10, IL12, IL17, IL18, IL1β, IFNγ, TNFα, TNFβ, TGFβ, and GM-CSF) were quantified upon stimulation of whole blood with latent state antigen EBNA1, lytic cycle antigen EBV-EA/D, and the superantigen SEB. To avoid results affected by lack of lymphocytes, we focused on SLE patients with normal levels. Decreased induction of IL12, IFNγ, IL17, and IL6 upon EBNA1 stimulation and that of IFNγ, IL6, TNFβ, IL1β, and GM-CSF upon EBV-EA/D stimulation were detected in SLE patients compared to HCs. IFNγ responses, especially, were shown to be reduced. Induction of several cytokines was furthermore impaired in SLE patients upon SEB stimulation, but no difference was observed in basic levels. Results substantiate the previously proposed impaired regulation of the immune response against latent and lytic cycle EBV infection in SLE patients without lymphopenia. Furthermore, results indicate general dysfunction of leukocytes and their cytokine regulations in SLE patients.

Epstein-Barr virus lytic reactivation regulation and its pathogenic role in carcinogenesis.

Epstein-Barr virus (EBV) has been associated with several types of human cancers. In the host, EBV can establish two alternative modes of life cycle, known as latent or lytic and the switch from latency to the lytic cycle is known as EBV reactivation. Although EBV in cancer cells is found mostly in latency, a small number of lytically-infected cells promote carcinogenesis through the release of growth factors and oncogenic cytokines. This review focuses on the mechanisms by which EBV reactivation is controlled by cellular and viral factors, and discusses how EBV lytic infection contributes to human malignancies.

Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells.

Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.

Production of thyrotropin receptor antibodies in acute phase of infectious mononucleosis due to Epstein-Barr virus primary infection: a case report of a child.

Various autoantibodies have been reported to be detected during the progression of infectious mononucleosis. We observed a case of infectious mononucleosis due to Epstein–Barr virus primary infection for 2 months, and noticed the transiently increased titer of thyrotropin receptor autoantibodies detected at the acute phase on the 3rd day after admission. At that time, real-time quantitative PCR also revealed the mRNA expressions of an immediate early lytic gene, BZLF1, and a latent gene, EBNA2. The expression of BZLF1 mRNA means that Epstein–Barr virus infects lytically, and EBNA2 protein has an important role in antibody production as well as the establishment of Epstein–Barr virus latency. These results suggest that Epstein–Barr virus lytic infection is relevant to thyrotropin receptor autoantibody production. Thyrotropin receptor autoantibodies stimulate thyroid follicular cells to produce excessive thyroid hormones and cause Graves’ disease. Recently, we reported the thyrotropin receptor autoantibody production from thyrotropin receptor autoantibody-predisposed Epstein–Barr virus-infected B cells by the induction of Epstein–Barr virus lytic infection in vitro. This case showed in vivo findings consistent with our previous reports, and is important to consider the pathophysiology of Graves’ disease and one of the mechanisms of autoimmunity.

Epstein–Barr virus BZLF1 protein impairs accumulation of host DNA damage proteins at damage sites in response to DNA damage

Epstein–Barr virus (EBV) infection is closely associated with several human malignancies including nasopharyngeal carcinoma (NPC). The EBV immediate-early protein BZLF1 is the key mediator that switches EBV infection from latent to lytic forms. The lytic form of EBV infection has been implicated in human carcinogenesis but its molecular mechanisms remain unclear. BZLF1 has been shown to be a binding partner of several DNA damage response (DDR) proteins. Its functions in host DDR remain unknown. Thus, we explore the effects of BZLF1 on cellular response to DNA damage in NPC cells. We found that expression of BZLF1 impaired the binding between RNF8 and MDC1 (mediator of DNA damage checkpoint 1), which in turn interfered with the localization of RNF8 and 53BP1 to the DNA damage sites. The RNF8-53BP1 pathway is important for repair of DNA double-strand breaks and DNA damage-induced G2/M checkpoint activation. Our results showed that, by impairing DNA damage repair as well as abrogating G2/M checkpoint, BZLF1 induced genomic instability and rendered cells more sensitive to ionizing radiation. Moreover, the blockage of 53BP1 and RNF8 foci formation was recapitulated in EBV-infected cells. Taken together, our study raises the possibility that, by causing mis-localization of important DDR proteins, BZLF1 may function as a link between lytic EBV infection and impaired DNA damage repair, thus contributing to the carcinogenesis of EBV-associated human epithelial malignancies.

Human Natural Killer Cells Prevent Infectious Mononucleosis Features by Targeting Lytic Epstein-Barr Virus Infection

Human Natural Killer Cells Prevent Infectious Mononucleosis Features by Targeting Lytic Epstein-Barr Virus Infection

Transforming growth factor beta 1 (TGF-β1) modulates Epstein-Barr virus reactivation in absence of Helicobacter pylori infection in patients with gastric cancer

BackgroundTransforming growth factor-beta 1 (TGF-β1), a multifunctional cytokine, acts as a key factor for Epstein-Barr virus (EBV) reactivation. We investigated the role of TGF-β1 in latent and lytic stages of EBV in relation to Helicobacter pylori infection among patients with gastric cancer (GC) and peptic ulcer disease (PUD). MethodGastric mucosal TGF-β1 expression was determined in 95 EBV positive patients with gastroduodenal pathology [GC 40, PUD 19 and non-ulcer dyspepsia (NUD) 36] by quantitative real time PCR. Presence of H. pylori infection was diagnosed when either culture or any two of three tests (RUT, histopathology and specific ureA PCR) were positive. Serum level of TGF-β1 was detected among 60 patients using ELISA. ResultsMucosal TGF-β1 mRNA expression was detected in 85 of 95 EBV positive patients and it was significantly higher in patients with GC (p=0.042). TGF-β1 expression tended to be higher among H. pylori non-infected than infected patients (3.80±6.24 vs. 2.07±2.50, p=0.085). Both mRNA and serum level had significant association with lytic stage of EBV in absence of H. pylori infection when compared with its presence (5.21±4.00 vs. 2.29±2.89, p=0.040 and 842.00 [669.55] vs. 662.63 [628.76], p=0.049; respectively). ConclusionTGF-β1 expression was significantly associated with GC. TGF-β1 was higher both at expression and translational levels in lytic EBV infection without H. pylori suggests that H. pylori infection might play important role in preventing EBV reactivation through attenuated TGF-β1 expression. This might be a “wise host defense against EBV reactivation”.

Isotypes of Epstein-Barr virus antibodies in rheumatoid arthritis: association with rheumatoid factors and citrulline-dependent antibodies.

In order to study the humoral immune response against Epstein-Barr virus (EBV) in patients with rheumatoid arthritis (RA) and to compare it with the two major autoantibody types in RA, plasma samples from 77 RA patients, 28 patients with systemic lupus erythematosus (SLE), and 28 healthy controls (HCs) were investigated by enzyme-linked immunosorbent assays (ELISA). Increased percentages of positives and concentrations of IgG/IgA/IgM antibodies against the latent EBV nuclear antigen-1 (EBNA-1) were observed in RA patients compared to SLE patients and HCs. Increased concentrations and percentages of positives of IgG/IgA/IgM against the early lytic EBV antigen diffuse (EAD) were also found in RA patients compared to HCs but were highest in SLE patients. Furthermore, associations between the elevated EBNA-1 IgA and EBNA-1 IgM levels and the presence of IgM and IgA rheumatoid factors (RFs) and anti-citrullinated protein antibodies (ACPAs, IgG) and between elevated IgA concentrations against EAD and the presence of RFs and ACPAs in RA patients were found. Thus, RA patients had elevated antibodies of all isotypes characteristic of latent EBV infection (whereas SLE patients had elevated antibodies characteristic of lytic EBV infection). Notably, for IgM and IgA (but not IgG), these were associated with the presence of characteristic RA autoantibodies.

Cellular differentiation regulator BLIMP1 induces Epstein-Barr virus lytic reactivation in epithelial and B cells by activating transcription from both the R and Z promoters.

Epstein-Barr virus (EBV) maintains a lifelong latent infection within a subset of its host's memory B cells, while lytic EBV replication takes place in plasma cells and differentiated epithelial cells. Therefore, cellular transcription factors, such as BLIMP1, that are key mediators of differentiation likely contribute to the EBV latent-to-lytic switch. Previous reports showed that ectopic BLIMP1 expression induces reactivation in some EBV-positive (EBV(+)) B-cell lines and transcription from Zp, with all Z(+) cells in oral hairy leukoplakia being BLIMP1(+). Here, we examined BLIMP1's role in inducing EBV lytic gene expression in numerous EBV(+) epithelial and B-cell lines and activating transcription from Rp. BLIMP1 addition was sufficient to induce reactivation in latently infected epithelial cells derived from gastric cancers, nasopharyngeal carcinomas, and normal oral keratinocytes (NOK) as well as some, but not all B-cell lines. BLIMP1 strongly induced transcription from Rp as well as Zp, with there being three or more synergistically acting BLIMP1-responsive elements (BRE) within Rp. BLIMP1's DNA-binding domain was required for reactivation, but BLIMP1 did not directly bind the nucleotide (nt) -660 Rp BRE. siRNA knockdown of BLIMP1 inhibited 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced lytic reactivation in NOK-Akata cells, cells that can be reactivated by R, but not Z. Thus, we conclude that BLIMP1 expression is both necessary and sufficient to induce EBV lytic replication in many (possibly all) EBV(+) epithelial-cell types, but in only a subset of EBV(+) B-cell types; it does so, at least in part, by strongly activating expression of both EBV immediately early genes, BZLF1 and BRLF1. This study is the first one to show that the cellular transcription factor BLIMP1, a key player in both epithelial and B-cell differentiation, induces reactivation of the oncogenic herpesvirus Epstein-Barr virus (EBV) out of latency into lytic replication in a variety of cancerous epithelial cell types as well as in some, but not all, B-cell types that contain this virus in a dormant state. The mechanism by which BLIMP1 does so involves strongly turning on expression of both of the immediate early genes of the virus, probably by directly acting upon the promoters as part of protein complexes or indirectly by altering the expression or activities of some cellular transcription factors and signaling pathways. The fact that EBV(+) cancers usually contain mostly undifferentiated cells may be due in part to these cells dying from lytic EBV infection when they differentiate and express wild-type BLIMP1.

Epstein-Barr virus infection and persistence in nasopharyngeal epithelial cells.

Epstein-Barr virus (EBV) infection is closely associated with undifferentiated nasopharyngeal carcinoma (NPC), strongly implicating a role for EBV in NPC pathogenesis; conversely, EBV infection is rarely detected in normal nasopharyngeal epithelial tissues. In general, EBV does not show a strong tropism for infecting human epithelial cells, and EBV infection in oropharyngeal epithelial cells is believed to be lytic in nature. To establish life-long infection in humans, EBV has evolved efficient strategies to infect B cells and hijack their cellular machinery for latent infection. Lytic EBV infection in oropharyngeal epithelial cells, though an infrequent event, is believed to be a major source of infectious EBV particles for salivary transmission. The biological events associated with nasopharyngeal epithelial cells are only beginning to be understood with the advancement of EBV infection methods and the availability of nasopharyngeal epithelial cell models for EBV infection studies. EBV infection in human epithelial cells is a highly inefficient process compared to that in B cells, which express the complement receptor type 2 (CR2) to mediate EBV infection. Although receptor(s) on the epithelial cell surface for EBV infection remain(s) to be identified, EBV infection in epithelial cells could be achieved via the interaction of glycoproteins on the viral envelope with surface integrins on epithelial cells, which might trigger membrane fusion to internalize EBV in cells. Normal nasopharyngeal epithelial cells are not permissive for latent EBV infection, and EBV infection in normal nasopharyngeal epithelial cells usually results in growth arrest. However, genetic alterations in premalignant nasopharyngeal epithelial cells, including p16 deletion and cyclin D1 overexpression, could override the growth inhibitory effect of EBV infection to support stable and latent EBV infection in nasopharyngeal epithelial cells. The EBV episome in NPC is clonal in nature, suggesting that NPC develops from a single EBV-infected nasopharyngeal epithelial cell, and the establishment of persistent and latent EBV infection in premalignant nasopharyngeal epithelium may represent an early and critical event for NPC development.

Implication of Epstein-Barr virus infection in disease-specific autoreactive B cell activation in ectopic lymphoid structures of Sjögren's syndrome.

To examine whether the B cell tropic γ-herpesvirus Epstein-Barr virus (EBV) is aberrantly expressed in its latent and lytic forms within ectopic lymphoid structures (ELS) in the salivary glands of patients with Sjögren's syndrome (SS), and to investigate the relationship between EBV dysregulation, B cell activation, in situ differentiation of autoreactive plasma cells, disease-specific autoantibody production, and cytotoxicity. Latent and lytic EBV infection in the salivary glands of 28 patients with SS and 38 patients with nonspecific chronic sialadenitis (NSCS), characterized for the presence or absence of ELS, was investigated by reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemical/immunofluorescence staining. Glandular versus synovial production of anti-Ro 52, anti-citrullinated protein antibodies (ACPAs), and anti-EBV peptide antibodies was analyzed in situ or in vivo in human SS/SCID and human rheumatoid arthritis/SCID mouse chimeras. EBV dysregulation within inflammatory infiltrates was observed exclusively in ELS+ SS salivary gland tissue, as revealed by latent EBV infection and lytic EBV infection in B cells and plasma cells, respectively. Conversely, epithelial latent membrane protein 2A expression was observed in both patients with SS and patients with NSCS. Importantly, perifollicular plasma cells displaying Ro 52 immunoreactivity were frequently infected by EBV. Furthermore, ELS-containing SS salivary gland tissue that was transplanted into SCID mice supported the production of anti-Ro 52/anti-La 48 and anti-EBV antibodies but not ACPAs. Analysis of CD4+ and CD8+ T cell localization and granzyme B expression demonstrated that the persistence of EBV in ELS-containing SS salivary glands was associated with follicular exclusion of CD8+ T cells and impaired CD8-mediated cytotoxicity. Active EBV infection is selectively associated with ELS in the salivary glands of patients with SS and appears to contribute to local growth and differentiation of disease-specific autoreactive B cells.

Quantitative detection of epstein-barr virus DNA in cerebrospinal fluid and blood samples of patients with relapsing-remitting multiple sclerosis.

The presence of Epstein-Barr Virus (EBV) DNA in cerebrospinal fluid (CSF) and peripheral blood (PB) samples collected from 55 patients with clinical and radiologically-active relapsing-remitting MS (RRMS) and 51 subjects with other neurological diseases was determined using standardized commercially available kits for viral nucleic acid extraction and quantitative EBV DNA detection. Both cell-free and cell-associated CSF and PB fractions were analyzed, to distinguish latent from lytic EBV infection. EBV DNA was detected in 5.5% and 18.2% of cell-free and cell-associated CSF fractions of patients with RRMS as compared to 7.8% and 7.8% of controls; plasma and peripheral blood mononuclear cells (PBMC) positivity rates were 7.3% and 47.3% versus 5.8% and 31.4%, respectively. No significant difference in median EBV viral loads of positive samples was found between RRMS and control patients in all tested samples. Absence of statistically significant differences in EBV positivity rates between RRMS and control patients, despite the use of highly sensitive standardized methods, points to the lack of association between EBV and MS disease activity.