Epstein-Barr virus (EBV) is a ubiquitous human pathogen that is transmitted in saliva. EBV transits through the oral epithelium to infect B cells, where it establishes a life-long latent infection. Reinfection of the epithelium is believed to be mediated by virus shed from B cells, but whether a latent reservoir can exist in the epithelia is unknown. We previously developed an in vitro organotypic model of stratified epithelium where EBV can readily replicate within the suprabasal layers of the epithelium following apical infection mediated by virus-producing B cells. Given that infected epithelial cells and cell-free virus are observed in saliva, we examined the ability of both of these to mediate infection in organotypic cultures. Epithelial-derived cell-free virus was able to infect organotypic cultures from the apical surface, but showed enhanced infection of B cells. Conversely, B cell-derived virus exhibited enhanced infection of epithelial cells. While EBV has been detected in basal cells in oral hairy leukoplakia, it is unknown whether EBV can be seen in undifferentiated primary keratinocytes in the basal layer. Undifferentiated epithelial cells expressed proposed EBV receptors in monolayer and were susceptible to viral binding and entry. Integrins, and occasionally ephrin A2, were expressed in the basal layer of gingiva and tonsil derived organotypic cultures, but the known B-cell receptors HLAII and CD21 were not detected. Following infection with cell-free virus or virus-producing B cells at either the apical or basolateral surface of preformed organotypic cultures, abundant infection was detected in differentiated suprabasal cells while more limited but readily detectable infection was observed in the undifferentiated basal cells. Together, our data has provided new insight into EBV infection in stratified epithelium.

The ubiquitous herpesvirus Epstein-Barr virus (EBV) is associated with cancers of B lymphocytes and epithelial cells and is primarily transmitted in saliva. While several models exist for analyzing the life cycle of EBV in B lymphocytes, models of EBV infection in the epithelium have more recently been established.

The ubiquitous and cancer-associated Epstein-Barr virus (EBV) is associated with nearly all cases of nasopharyngeal carcinoma (NPC). Nasopharyngeal tissue is comprised of both pseudostratified and stratified epithelium, which are modeled in three-dimensional (3-D) cell culture. The cellular origin of EBV-associated NPC is as yet unknown, but both latent and lytic infections are likely important for preneoplastic mechanisms and replenishing the compartmentalized viral reservoir. Conventional 2-D cultures of nasopharyngeal epithelial cells (as primary cells or immortalized cell lines) are difficult to infect with EBV and cannot mimic the tissue-specific biology of the airway epithelium, which can only be captured in 3-D models. We have shown that EBV can infect the pseudostratified epithelium in air-liquid interface (ALI) culture using primary conditionally reprogrammed cells (CRCs) derived from the nasopharynx. In this protocol, we provide a step-by-step guide for the (i) conditional reprogramming of primary nasopharyngeal cells, (ii) differentiation of CRCs into pseudostratified epithelium in ALI culture (known as pseudo-ALI), and (iii) EBV infection of pseudo-ALI cultures. Additionally, we show that nasopharyngeal CRCs can be grown as organotypic rafts and subjected to EBV infection. These nasopharyngeal-derived 3-D cell cultures can be used to study EBV latent and lytic infection in relation to cell type and donor variation, by immunostaining and single-cell RNA-sequencing methods (Ziegler et al., 2021). These methods are useful for studies of EBV molecular pathogenesis, and can overcome many of the limitations associated with conventional 2-D cell cultures.Graphic abstract: Workflow of nasopharyngeal-derived conditionally reprogrammed cells grown into pseudostratified-ALI and organotypic rafts in 3-D cell culture. Created with Biorender.com.

Epstein-Barr Virus (EBV) is a γ-herpesvirus which infects over 90% of the adult human population. Most notably, this virus causes infectious mononucleosis but it is also associated with cancers such as Hodgkin and Burkitt lymphoma. EBV is a species-specific virus and has been studied in many animal models, including nonhuman primates, guinea pigs, humanized mice, and tree shrews. However, none of these animal models are considered the "gold standard" for EBV research. Recently, rabbits have emerged as a viable alternative model, as they are susceptible to EBV infection. In addition, the EBV infection progresses after immune suppression with cyclosporine A (CsA), modeling the reactivation of EBV after latency. We sought to refine this model for acute or active EBV infections by performing antibody-mediated depletion of certain immune subsets in rabbits. Fourteen 16 to 20-wk old, NZW rabbits were intravenously inoculated with EBV and concurrently treated with either anti-CD4 T-cell antibody, anti-pan-T-cell antibody (anti CD45), CSA, or, as a control, anti-HPV antibody. Rabbits that received the depleting antibodies were treated with CsA 3 times at a dose of 15 mg/kg SC once per day for 4 d starting at the time of EBV inoculation then the dose was increased to 20 mg/kg SC twice weekly for 2 wk. Weights, temperatures, and clinical signs were monitored, and rabbits were anesthetized once weekly for blood collection. When compared with the control group, anti-CD4-treated rabbits had fewer clinical signs and displayed higher levels of viral DNA via qPCR in splenocytes; however, flow cytometry results showed only a partial depletion of CD4 T-cells. Treatment with anti-pan-T-cell antibody did not result in noticeable T-cell depletion. These data suggest the EBV-infected rabbit is a promising model for testing antiviral medications and prophylactic vaccines for EBV.

While numerous model systems are available to study EBV latency in B cells and have contributed greatly to our understanding of the role of these cells in the viral life cycle, models to study the EBV life cycle in epithelial cells in vitro are lacking. Epithelial cells are poorly infected in vitro, and EBV-infected cell lines have not been successfully obtained from epithelial tumors. Recently, we have demonstrated that organotypic cultures of oral keratinocytes can be used as a model to study EBV infection in the epithelial tissue. These "raft" cultures generate a stratified tissue resembling the epithelium seen in vivo with a proliferating basal layer and differentiating suprabasal layers. Here, we describe generation of EBV-infected raft cultures established from primary oral mucosal epithelial cells, which exhibit high levels of productive replication induced by differentiation, as well as methods to analyze EBV infection.

With an increasing number of clinical trials looking at combination therapies in cancer, potential drug-drug interactions require particular attention. One such instance is the treatment of CD30(+) tumors after previous vorinostat (SAHA; suberoylanilide hydroxyamic acid) failure with the anti-CD30 antibody-drug conjugate brentuximab vedotin. Using B-, T-, and natural killer (NK)-cell lines in vitro, we demonstrate that SAHA downregulates the expression of CD30 and lowers the efficacy of subsequent brentuximab vedotin treatment if baseline CD30 levels are reduced by 50% or more. Interestingly, low-dose SAHA treatment that maintained 50% or more of basal CD30 expression followed by subsequent treatment with brentuximab vedotin led to enhanced antitumor activity. The downregulation of CD30 was short lived upon SAHA removal, suggesting that allowing SAHA washout may circumvent any interactions with subsequent drug therapies. Our findings confirm the requirement of CD30 for brentuximab vedotin efficacy and suggest that combination treatment with SAHA in CD30(dim) tumors may decrease efficacy. Combination treatment in highly CD30(+) tumors, however, increases efficacy and warrants further consideration as a new treatment paradigm.

Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF) and p16(INK4a) expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF) and p16I(NK4a). By contrast, p16(INK4a) was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF) was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1), a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1) expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.

Epstein-Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi's-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.

ABSTRACT An ordered silencing of Epstein-Barr virus (EBV) latency gene transcription is critical for establishment of persistent infection within B lymphocytes, yet the mechanisms responsible and the role that the virus itself may play are unclear. Here we describe two B-cell superinfection models with which to address these problems. In the first, Burkitt lymphoma (BL) cells that maintain latency I, when superinfected, initially supported transcription from the common EBNA promoters Wp and Cp (latency III) but ultimately transitioned to latency I (Cp/Wp silent), an essential requirement for establishment of EBV latency in vivo. We used this model to test whether the early lytic-cycle gene BHLF1, implicated in silencing of the Cp/Wp locus, is required to establish latency I. Upon superinfection with EBV deleted for the BHLF1 locus, however, we have demonstrated that BHLF1 is not essential for this aspect of EBV latency. In the second model, BL cells that maintain Wp-restricted latency, a variant program in which Cp is silent but Wp remains active, sustained the latency III program of transcription from the superinfecting-virus genomes, failing to transition to latency I. Importantly, there was substantial reduction in Wp-mediated protein expression from endogenous EBV genomes, in the absence of Cp reactivation, that could occur independent of a parallel decrease in mRNA. Thus, our data provide evidence of a novel, potentially posttranscriptional mechanism for trans-repression of Wp-dependent gene expression. We suggest that this may ensure against overexpression of the EBV nuclear antigens (EBNAs) prior to the transcriptional repression of Wp in cis that occurs upon activation of Cp.

Establishment of persistent Epstein-Barr virus (EBV) infection requires transition from a program of full viral latency gene expression (latency III) to one that is highly restricted (latency I and 0) within memory B lymphocytes. It is well established that DNA methylation plays a critical role in EBV gene silencing, and recently the chromatin boundary protein CTCF has been implicated as a pivotal regulator of latency via its binding to several loci within the EBV genome. One notable site is upstream of the common EBNA gene promoter Cp, at which CTCF may act as an enhancer-blocking factor to initiate and maintain silencing of EBNA gene transcription. It was previously suggested that increased expression of CTCF may underlie its potential to promote restricted latency, and here we also noted elevated levels of DNA methyltransferase 1 (DNMT1) and DNMT3B associated with latency I. Within B-cell lines that maintain latency I, however, stable knockdown of CTCF, DNMT1, or DNMT3B or of DNMT1 and DNMT3B in combination did not result in activation of latency III protein expression or EBNA gene transcription, nor did knockdown of DNMTs significantly alter CpG methylation within Cp. Thus, differential expression of CTCF and DNMT1 and -3B is not critical for maintenance of restricted latency. Finally, mutant EBV lacking the Cp CTCF binding site exhibited sustained Cp activity relative to wild-type EBV in a recently developed B-cell superinfection model but ultimately was able to transition to latency I, suggesting that CTCF contributes to but is not necessarily essential for the establishment of restricted latency.

Epstein–Barr virus (EBV) is a potent transformer of B lymphocytes in vitro and is associated with a half dozen human cancers. Yet, while 90% of the world population is persistently infected with this herpesvirus, the overall incidence of EBV-related malignancy is exceedingly low. This reflects the highly evolved and finely balanced relationship between EBV and its host. Viral persistence is mediated through a small subset of the more than 80 genes encoded by EBV, the majority of which contribute to virus replication and are silent during latent infection. The latency-associated proteins are a six-member family of EBV nuclear proteins (EBNAs), three integral membrane proteins (LMPs), and several small proteins potentially expressed from a family of alternatively spliced RNAs (BARTs). In addition, two small noncoding RNAs (EBERs) and multiple micro-RNAs are expressed during latency. Known functions of the latency-associated gene products range from viral genome maintenance, regulation of EBV and cellular gene transcription, promotion of cell survival and proliferation, to evasion of host immune-response mechanisms. Selective expression of the EBV latency genes within several distinct latency programs promotes the establishment and maintenance of a persistent infection, while ensuring that the oncogenic potential of the virus is limited.

EBV-associated malignancies remain a considerable problem in HIV-infected individuals, even in the era of HAART. Although EBV is a common factor, each disease has a unique pathogenesis. Study of these diseases reveals the viral proteins expressed in the malignancies that might contribute to the development of the disease as well as the molecular basis for pathogenesis. It is likely that this knowledge will contribute to the development of novel therapeutics that will result in more favorable outcomes in the future.

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is essential for EBV-mediated immortalization of human B lymphocytes and regulates both the cell cycle and transcription. Transient reporter gene assays have implicated a pivotal role for EBNA-3C in the regulation of transcription of the majority of latency-associated genes expressed during the EBV growth program, including the viral oncoprotein LMP-1. To examine the regulation of latency gene expression by EBNA-3C, we generated an EBV-positive cell line that inducibly expresses EBNA-3C. This cell line allowed us to examine expression from the endogenous latency gene promoters in the context of an actual latent infection and the presence of other EBNA proteins, in particular EBNA-2, which is presumed to coregulate transcription with EBNA-3C. EBNA-3C induced the expression of both LMP-1 and LMP-2B mRNAs from the bidirectional LMP-1/LMP-2B promoter. In contrast, no effect was seen on expression from the common EBNA promoter Cp, which is responsive to EBNA-3C in reporter assays. Activation of LMP expression was not the consequence of increases in EBNA-2, PU.1 or Spi-B transcription factors, all of which are believed to be critical for activation of LMP-1. Chromatin immunoprecipitation assays furthermore indicated that EBNA-3C is present at the bidirectional LMP-1/LMP-2B promoter. These results indicate that EBNA-3C directly activates the expression of LMP-1 and LMP-2B but is unlikely to significantly regulate EBNA expression via Cp under normal growth conditions.

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is a large transcriptional regulator essential for EBV-mediated immortalization of B lymphocytes. We previously identified interactions between EBNA-3C and two cellular transcription factors, Jκ and Spi proteins, through which EBNA-3C regulates transcription. To better understand the contribution of these interactions to EBNA-3C function and EBV latency, we examined whether they are conserved in the homologous proteins of nonhuman primate lymphocryptoviruses (LCVs), which bear a strong genetic and biological similarity to EBV. The homologue of EBNA-3C encoded by the LCV that infects baboons (BaLCV) was found to be only 35% identical in sequence to its EBV counterpart. Of particular significance, this homology localized predominantly to the N-terminal half of the molecule, which encompasses the domains in EBNA-3C that interact with Jκ and Spi proteins. Like EBNA-3C, both BaLCV and rhesus macaque LCV (RhLCV) 3C proteins bound to Jκ and repressed transcription mediated by EBNA-2 through its interaction with Jκ. Both nonhuman primate 3C proteins were also able to activate transcription mediated by the Spi proteins in the presence of EBNA-2. Like EBNA-3C, a domain encompassing the putative basic leucine zipper motif of the BaLCV-3C protein directly interacted with both Spi-1 and Spi-B. Surprisingly, a recently identified motif in EBNA-3C that mediates repression was not identifiable in the BaLCV-3C protein. Finally, although the C terminus of BaLCV-3C bears minimal homology to EBNA-3C, it nonetheless contains a C-terminal domain rich in glutamine and proline that was able to function as a potent transcriptional activation domain, as does the C terminus of EBNA-3C. The conservation of these functional motifs despite poor overall homology among the LCV 3C proteins strongly suggests that the interactions of EBNA-3C with Jκ and Spi do indeed play significant roles in the life cycle of EBV.

Viruses utilize numerous mechanisms to counteract the host's immune response. Interferon production is a major component of the host antiviral response. Many viruses, therefore, produce proteins or RNA molecules that inhibit interferon-induced signal transduction pathways and their associated antiviral effects. Surprisingly, some viruses directly induce expression of interferon-induced genes. SM, an early lytic Epstein-Barr virus (EBV) nuclear protein, was found to specifically increase the expression of several genes (interferon-stimulated genes) that are known to be strongly induced by alpha/beta interferons. SM does not directly stimulate alpha/beta interferon secretion but instead induces STAT1, an intermediate step in the interferon signaling pathway. SM is a posttranscriptional activator of gene expression and increases STAT1 mRNA accumulation, particularly that of the functionally distinct STAT1beta splice variant. SM expression in B lymphocytes is associated with decreased cell proliferation but does not decrease cell viability or induce cell cycle arrest. These results indicate that EBV can specifically induce cellular genes that are normally physiological targets of interferon by inducing components of cytokine signaling pathways. Our findings therefore suggest that some aspects of the interferon response may be positively modulated by infecting viruses.

Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor J kappa, This interaction downregulates transcription mediated by EBNA-2 and J kappa. TO identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with J kappa, Using a reporter gene assay based on the recruitment of J kappa by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of J kappa to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with J kappa. To determine the biological significance of the interaction of EBNA-3A with J kappa, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with J kappa through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and J kappa, The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes.

There is a growing interest in using antigen-specific T cells for the treatment of human malignancy. For example, adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has been effective prophylaxis and treatment of EBV-associated lymphoproliferative disease in immunocompromised patients. For all immunotherapies, however, there has been a hypothetical concern that mutations in tumor-specific antigens may lead to tumor escape. We now demonstrate that such events may indeed occur, with lethal outcome. A patient who developed lymphoma after marrow transplantation received donor-derived, EBV-specific CTLs but died with progressive disease. The tumor cells proved substantially less sensitive to cytolysis than the EBV-transformed B-cell line used for CTL generation. The major cytolytic activity of the donor CTL was directed against 2 HLA-A11-restricted epitopes in the viral EBNA-3B antigen. Sequence analysis of this gene in the tumor virus revealed a 245-base pair deletion, which removed these 2 CTL epitopes. Hence, the viral antigen in the tumor had mutated in a way that allowed escape from CTLs. Analysis of EBV polymorphisms demonstrated that before CTL infusion, more than one virus was present, including a virus with wild-type EBNA-3B. After CTL infusion, only the virus with the EBNA-3B deletion could be detected, suggesting that the infused CTLs had selected a resistant strain in vivo. Such an occurrence, even when polyclonal CTL lines are used against genetically stable virus antigens, suggests that escape mutants may be a serious problem when CTL therapy is directed against more unstable tumor cell-derived targets.

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) protein is a transcriptional regulator of viral and cellular genes that is essential for EBV-mediated immortalization of B lymphocytes in vitro. EBNA-3C can inhibit transcription through an association with the cellular DNA-binding protein Jκ, a function shared by EBNA-3A and EBNA-3B. Here, we report a mechanism by which EBNA-3C can activate transcription from the EBV latent membrane protein 1 (LMP-1) promoter in conjunction with EBNA-2. Jκ DNA-binding sites were not required for this activation, and a mutant EBNA-3C protein unable to bind Jκ activated transcription as efficiently as wild-type EBNA-3C, indicating that EBNA-3C can regulate transcription through a mechanism that is independent of Jκ. Furthermore, activation of the LMP-1 promoter is a unique function of EBNA-3C, not shared by EBNA-3A and EBNA-3B. The DNA element through which EBNA-3C activates the LMP-1 promoter includes a Spi-1/Spi-B binding site, previously characterized as an important EBNA-2 response element. Although this element has considerable homology to mouse immunoglobulin light chain promoter sequences to which the mouse homologue of Spi-1 binds with its dimerization partner IRF4, we demonstrate that the IRF4-like binding sites in the LMP-1 promoter do not play a role in EBNA-3C-mediated activation. Both EBNA-2 and EBNA-3C were required for transcription mediated through a 41-bp region of the LMP-1 promoter encompassing the Spi binding site. However, EBNA-3C had no effect on transcription mediated in conjunction with the EBNA-2 activation domain fused to the GAL4 DNA-binding domain, suggesting that it does not function as an adapter between EBNA-2 and the cellular transcriptional machinery. Like EBNA-2, EBNA-3C bound directly to both Spi-1 and Spi-B in vitro. This interaction was mediated by a region of EBNA-3C encompassing a likely basic leucine zipper (bZIP) domain and the ets domain of Spi-1 or Spi-B, reminiscent of interactions between bZIP and ets domains of other transcription factors that result in their targeting to DNA. There are many examples of regulation of the hematopoietic-specific Spi transcription factors through protein-protein interactions, and a similar regulation by EBNA-3C, in conjunction with EBNA-2, is likely to be an important and unique contribution of EBNA-3C to EBV-mediated immortalization.