Epstein-Barr virus infection in the development of neurological disorders

Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.

Lymphoproliferation in children after liver transplantation.

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.

Epstein–Barr Virus Infection Induces Aberrant TLR Activation Pathway and Fibroblast–Myofibroblast Conversion in Scleroderma

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.

Distribution and Phenotype of Epstein-Barr Virus-Infected Cells in Inflammatory Bowel Disease

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.

Poorly or undifferentiated nasopharyngeal carcinoma (NPC) is regularly associated with Epstein-Barr virus (EBV) infection. EBV infection of premalignant nasopharyngeal epithelial cells has been postulated to play an important role in the pathogenesis of nasopharyngeal carcinoma. We have previously reported that genetic alterations could be detected in premalignant nasopharyngeal epithelial tissues. It is not clear if these genetic alterations may associate with the establishment of EBV infection. In this study, we have examined the role of cyclin D1 in supporting EBV infection in nasopharyngeal epithelial cells. While cyclin D1 is commonly expressed in nasopharyngeal carcinoma tissues we found that overexpression of cycline D1 is readily detected in dysplastic nasopharyngeal epithelial closely associated with EBV infection. Furthermore, using a panel of immortalized nasopharyngeal epithelial cells, we demonstrated that overexpression of cyclin D1 or CKD4 supports EBV infection and result in the establishment of stable EBV infected nasopharyngeal cell lines. We also observed that EBV infection induced growth inhibition and senescence in nasopharyngeal epithelial cells, which may be the underlying reason for low detection rate of EBV infection in healthy nasopharyngeal epithelial tissues. However, cyclin D1 overexpression suppressed senescence and differentiation in the immortalized nasopharyngeal epithelial cells, suggesting cyclin D1 may play an important role in supporting EBV infection in these cells. We postulated that the altered expression of cyclin D1 or CDK4 in premalignant nasopharyngeal epithelial cells may over-ride the growth suppression effects of EBV infection and support EBV latent infection in premalignant nasopharyngeal epithelial cells. Acknowledgement: This study is sponsored by grant support received from the Hong Kong Research Grant Council (Grant number: GRF780911 and AoE /M-06/08) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2037. doi:1538-7445.AM2012-2037

Latent Epstein-Barr virus (EBV) infection promotes undifferentiated nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC), while EBV infection of normal differentiated oropharyngeal epithelial cells is lytic and kills the cell. Establishment of viral latency within epithelial cells is likely essential for the development of EBV-induced NPCs and GCs, but the mechanism(s) by which EBV latency is maintained in epithelial cells are not fully understood. Here we demonstrate that the cellular tumor suppressor protein IRF6, a master regulator of squamous cell epithelial cell differentiation, plays a critical role in promoting TPA-induced lytic EBV reactivation in vitro in both EBV-infected NPC cells and EBV-infected GC cells. Using a telomerase-immortalized normal oral keratinocyte cell line (NOKs) model which retains the ability to differentiate in response to TPA treatment, we show that TPA-induced lytic EBV reactivation requires the PKCδ-RIPK4-IRF6 signaling pathway. RIPK4 is a PKCδ (PRKCD)-activated cellular S/T kinase that phosphorylates and activates the IRF6 transcription factor. We demonstrate that inhibition of PKCδ, RIPK4 or IRF6 expression is sufficient to suppress TPA-induced epithelial cell differentiation, as well as lytic EBV reactivation, in NOKs. Furthermore, we find that latent EBV infection in NOKs inhibits the expression of IRF6. Importantly, we show that inducible expression of a constitutively active (phospho-mimetic) IRF6 mutant is sufficient to activate the lytic form of EBV infection in both EBV-infected NOKs and EBV-infected SNU719 GC cells. Finally, we demonstrate that the ability of constitutively active IRF6 to promote lytic EBV infection in NOKs is at least partially mediated by IRF6-induced expression of the BLIMP1 transcription factor, which we previously showed synergistically activates expression of the two EBV immediate-early proteins, BZLF1 and BRLF1, in conjunction with KLF4. Thus, suppression of IRF6 expression may promote NPC and GC tumors by blocking lytic EBV reactivation and differentiation.

SP0133 Raise for Impact: Epstein-Barr Virus in Rheumatic Patients

An amplifying role for oral epithelial cells (ECs) in Epstein-Barr Virus (EBV) infection has been postulated to explain oral viral shedding. However, while lytic or latent EBV infections of oro/nasopharyngeal ECs are commonly detected under pathological conditions, detection of EBV-infected ECs in healthy conditions is very rare. In this study, a simple non-surgical tissue sampling procedure was used to investigate EBV infection in the periodontal epithelium that surrounds and attaches teeth to the gingiva. Surprisingly, we observed that the gingival ECs of the periodontium (pECs) are commonly infected with EBV and may serve as an important oral reservoir of latently EBV-infected cells. We also found that the basal level of epithelial EBV-infection is significantly increased in chronic periodontitis, a common inflammatory disease that undermines the integrity of tooth-supporting tissues. Moreover, the level of EBV infection was found to correlate with disease severity. In inflamed tissues, EBV-infected pECs appear to be prone to apoptosis and to produce larger amounts of CCL20, a pivotal inflammatory chemokine that controls tissue infiltration by immune cells. Our discovery that the periodontal epithelium is a major site of latent EBV infection sheds a new light on EBV persistence in healthy carriers and on the role of this ubiquitous virus in periodontitis. Moreover, the identification of this easily accessible site of latent infection may encourage new approaches to investigate and monitor other EBV-associated disorders.

Most nasopharyngeal carcinoma (NPC) biopsies contain a human pathogen named as Epstein-Barr virus (EBV). EBV infection is considered as an early event involved in the pathogenesis of NPC. However, the close association of EBV with NPC is a paradox. EBV infection is common that occurs in ≥95% of the world's population, yet the incidence rate of NPC is rare worldwide and is only seen in Cantonese speaking ethnic groups in Southern Asia. The mechanisms that regulate EBV infection of NPC/pre-malignant NP cells including the events involved in latency and lytic activation (either in vitro or in vivo) are not well-defined. Expressions of latent and lytic EBV genes have been shown to have variable effects on the biology and malignant properties of EBV-infected cells. Hypoxia and nutrient-starvation are common physiological stresses within tumours and may have impact on regulation of latent and lytic EBV infection in NPC cells. Here we report that the levels of hypoxia-inducible factor, HIF-1α protein, a prime hypoxic-stress responsive marker for mammalian cells, are higher in EBV-infected NPC/pre-malignant NP cell lines under hypoxia than their non-infected counterparts, which may imply an enhanced survival ability of EBV-infected NP cells. The alteration in gene expression profiles of host cells and expression of EBV genes observed in infected cells may be responsible for the stabilization of HIF-1α and vice versa. Moreover, EBV infection enhances the tumour growth rate and size of NPC cells xenografts in nude mice. The main stem of this study is to examine whether there is a differential response between EBV-infected and non-infected NP cells towards various metabolic stresses. Through studying this project, the mechanism and function of HIF-1α stability by EBV infection will be explored, which is expected to help characterizing the role of EBV in transformation of epithelial cells and how EBV infection may enhance NPC cells to survive inside a tumour core. Citation Format: VMY Lau, SW Tsao. Role of Epstein-Barr virus in nasopharyngeal carcinoma under metabolic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1512. doi:10.1158/1538-7445.AM2013-1512 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Epstein-Barr Virus and Human Disease • 1988

Integrative Identification of Epstein–Barr Virus–Associated Mutations and Epigenetic Alterations in Gastric Cancer

Epstein-Barr virus (EBV) is a human herpesvirus that is associated with lymphomas as well as nasopharyngeal and gastric carcinomas. Although carcinomas account for almost 90% of EBV-associated cancers, progress in examining EBV's role in their pathogenesis has been limited by difficulty in establishing latent infection in nontransformed epithelial cells. Recently, EBV infection of human telomerase reverse transcriptase (hTERT)-immortalized normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection in vivo, such as differentiation-associated viral replication. Using uninfected NOKs and NOKs infected with the Akata strain of EBV (NOKs-Akata), we examined changes in gene expression due to EBV infection and differentiation. Latent EBV infection produced very few significant gene expression changes in undifferentiated NOKs but significantly reduced the extent of differentiation-induced gene expression changes. Gene set enrichment analysis revealed that differentiation-induced downregulation of the cell cycle and metabolism pathways was markedly attenuated in NOKs-Akata relative to that in uninfected NOKs. We also observed that pathways induced by differentiation were less upregulated in NOKs-Akata. We observed decreased differentiation markers and increased suprabasal MCM7 expression in NOKs-Akata versus NOKs when both were grown in raft cultures, consistent with our transcriptome sequencing (RNA-seq) results. These effects were also observed in NOKs infected with a replication-defective EBV mutant (AkataΔRZ), implicating mechanisms other than lytic-gene-induced host shutoff. Our results help to define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers.IMPORTANCE Latent infection by Epstein-Barr virus (EBV) is an early event in the development of EBV-associated carcinomas. In oral epithelial tissues, EBV establishes a lytic infection of differentiated epithelial cells to facilitate the spread of the virus to new hosts. Because of limitations in existing model systems, the effects of latent EBV infection on undifferentiated and differentiating epithelial cells are poorly understood. Here, we characterize latent infection of an hTERT-immortalized oral epithelial cell line (NOKs). We find that although EBV expresses a latency pattern similar to that seen in EBV-associated carcinomas, infection of undifferentiated NOKs results in differential expression of a small number of host genes. In differentiating NOKs, however, EBV has a more substantial effect, reducing the extent of differentiation and delaying the exit from the cell cycle. This effect may synergize with preexisting cellular abnormalities to prevent exit from the cell cycle, representing a critical step in the development of cancer.