Landscape of Epstein-Barr virus gene expression and perturbations in cancer.

Epstein-Barr virus (EBV) is the causative agent for multiple neoplastic diseases of epithelial and lymphocytic origin1-3. The heterogeneity of the viral elements expressed and the mechanisms by which these coding and non-coding genes maintain cancer cell properties in vivo remain elusive4,5. Here we conducted a multi-modal transcriptomic analysis of EBV-associated neoplasms and identified that the ubiquitously expressed RPMS1 non-coding RNAs support cancer cell properties by disruption of the interferon response. Our map of EBV expression shows a variable, but pervasive expression of BNLF2 discerned from the overlapping LMP1 RNA in bulk sequencing data. Using long-read single-molecule sequencing, we identified three new viral elements within the RPMS1 gene. Furthermore, single-cell sequencing datasets allowed for the separation of cancer cells and healthy cells from the same tissue biopsy and the characterization of a microenvironment containing interferon gamma excreted by EBV-stimulated T-lymphocytes. In comparison with healthy epithelium, EBV-transformed cancer cells exhibited increased proliferation and inhibited immune response induced by the RPMS1-encoded microRNAs. Our atlas of EBV expression shows that the EBV-transformed cancer cells express high levels of non-coding RNAs originating from RPMS1 and that the oncogenic properties are maintained by RPMS1 microRNAs. Through bioinformatic disentanglement of single cells from cancer tissues we identified a positive feedback loop where EBV-activated immune cells stimulate cancer cells to proliferate, which in turn undergo viral reactivation and trigger an immune response.

Serologic Profiling Using an Epstein-Barr Virus Mammalian Expression Library Identifies EBNA1 IgA as a Prediagnostic Marker for Nasopharyngeal Carcinoma.

PURPOSE: The favorable prognosis of stage I and II nasopharyngeal carcinoma (NPC) has motivated a search for biomarkers for the early detection and risk assessment of Epstein-Barr virus (EBV)-associated NPC. Although EBV seropositivity is ubiquitous among adults, a spike in antibodies against select EBV proteins is a harbinger of NPC. A serologic survey would likely reveal which EBV antibodies could discriminate those at risk of developing NPC. EXPERIMENTAL DESIGN: Lysates from a new EBV mammalian expression library were used in a denaturing multiplex immunoblot assay to survey antibodies against EBV in sera collected from healthy individuals who later developed NPC (incident cases) in a prospective cohort from Singapore and validated in an independent cohort from Shanghai, P.R. China. RESULTS: We show that IgA against EBV nuclear antigen 1 (EBNA1) discriminated incident NPC cases from matched controls with 100% sensitivity and 100% specificity up to 4 years before diagnosis in both Singapore and Shanghai cohorts. Incident NPC cases had a greater IgG repertoire against lytic-classified EBV proteins, and the assortment of IgA against EBV proteins detected by the immunoblot assay increased closer to diagnosis. CONCLUSIONS: Although NPC tumors consistently harbor latent EBV, the observed heightened systemic and mucosal immunity against lytic-classified antigens years prior to clinical diagnosis is consistent with enhanced lytic transcription. We conclude that an expanding EBV mucosal reservoir (which can be latent and/or lytic) is a risk factor for NPC. This presents an opportunity to identify those at risk of developing NPC using IgA against EBNA1 as a biomarker.

Three-dimensional Models of the Nasopharynx for the Study of Epstein-Barr Virus Infection.

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.

A primary nasopharyngeal three-dimensional air-liquid interface cell culture model of the pseudostratified epithelium reveals differential donor- and cell type-specific susceptibility to Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, α-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (~0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC.

Viral cGAMP nuclease reveals the essential role of DNA sensing in protection against acute lethal virus infection.

Cells contain numerous immune sensors to detect virus infection. The cyclic GMP-AMP (cGAMP) synthase (cGAS) recognizes cytosolic DNA and activates innate immune responses via stimulator of interferon genes (STING), but the impact of DNA sensing pathways on host protective responses has not been fully defined. We demonstrate that cGAS/STING activation is required to resist lethal poxvirus infection. We identified viral Schlafen (vSlfn) as the main STING inhibitor, and ectromelia virus was severely attenuated in the absence of vSlfn. Both vSlfn-mediated virulence and STING inhibitory activity were mapped to the recently discovered poxin cGAMP nuclease domain. Animals were protected from subcutaneous, respiratory, and intravenous infection in the absence of vSlfn, and interferon was the main antiviral protective mechanism controlled by the DNA sensing pathway. Our findings support the idea that manipulation of DNA sensing is an efficient therapeutic strategy in diseases triggered by viral infection or tissue damage-mediated release of self-DNA.

Circular DNA tumor viruses make circular RNAs.

Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) cause ∼2% of all human cancers. RNase R-resistant RNA sequencing revealed that both gammaherpesviruses encode multiple, uniquely stable, circular RNAs (circRNA). EBV abundantly expressed both exon-only and exon-intron circRNAs from the BamHI A rightward transcript (BART) locus (circBARTs) formed from a spliced BART transcript and excluding the EBV miRNA region. The circBARTs were expressed in all verified EBV latency types, including EBV-positive posttransplant lymphoproliferative disease, Burkitt lymphoma, nasopharyngeal carcinoma, and AIDS-associated lymphoma tissues and cell lines. Only cells infected with the B95-8 EBV strain, with a 12-kb BART locus deletion, were negative for EBV circBARTs. Less abundant levels of EBV circRNAs originating from LMP2- and BHLF1-encoding genes were also identified. The circRNA sequencing of KSHV-infected primary effusion lymphoma cells revealed a KSHV-encoded circRNA from the vIRF4 locus (circvIRF4) that was constitutively expressed. In addition, KSHV polyadenylated nuclear (PAN) RNA locus generated a swarm (>100) of multiply backspliced, low-abundance RNase R-resistant circRNAs originating in both sense and antisense directions consistent with a novel hyperbacksplicing mechanism. In EBV and KSHV coinfected cells, exon-only EBV circBARTs were located more in the cytoplasm, whereas the intron-retaining circBARTs were found in the nuclear fraction. KSHV circvIRF4 and circPANs were detected in both nuclear and cytoplasmic fractions. Among viral circRNAs tested, none were found in polysome fractions from KSHV-EBV coinfected BC1 cells, although low-abundance protein translation from viral circRNAs could not be excluded. The circRNAs are a new class of viral transcripts expressed in gammaherpesvirus-related tumors that might contribute to viral oncogenesis.

Air-Liquid Interface Method To Study Epstein-Barr Virus Pathogenesis in Nasopharyngeal Epithelial Cells.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes a latent reservoir in peripheral B-lymphocytes with sporadic reactivation. EBV also infects epithelial cells, predominantly resulting in a lytic infection, which may contribute to EBV transmission from saliva. In the nasopharynx, EBV infection can lead to the clonal expansion of a latently infected cell and the development of nasopharyngeal carcinoma (NPC). The mechanisms governing EBV pathogenesis in nasopharyngeal epithelium are largely unknown. An advanced understanding would depend on a physiologically relevant culture model of polarized airway epithelium. The recent application of the organotypic raft culture in keratinocytes has demonstrated great promise for the use of polarized cultures in the study of EBV permissive replication. In this study, the adaptation of an air-liquid interface (ALI) culture method using transwell membranes was explored in an EBV-infected NPC cell line. In the EBV-infected NPC HK1 cell line, ALI culture resulted in the completion of EBV reactivation, with global induction of the lytic cascade, replication of EBV genomes, and production of infectious progeny virus. We propose that the ALI culture method can be widely adopted as a physiologically relevant model to study EBV pathogenesis in polarized nasal epithelial cells. IMPORTANCE: Lifting adherent cells to the air-liquid interface (ALI) is a method conventionally used to culture airway epithelial cells into polarized apical and basolateral surfaces. Reactivation of Epstein-Barr virus (EBV) from monolayer epithelial cultures is sometimes abortive, which may be attributed to the lack of authentic reactivation triggers that occur in stratified epithelium in vivo In the present work, the ALI culture method was applied to study EBV reactivation in nasopharyngeal epithelial cells. The ALI culture of an EBV-infected cell line yielded high titers and can be dissected by a variety of molecular virology assays that measure induction of the EBV lytic cascade and EBV genome replication and assembly. EBV infection of polarized cultures of primary epithelial cells can be challenging and can have variable efficiencies. However, the use of the ALI method with established EBV-infected cell lines offers a readily available and reproducible approach for the study of EBV permissive replication in polarized epithelia.

Modified Anoikis Assay That Functionally Segregates Epstein-Barr Virus LMP1 Strains into Two Groups.

Nasopharyngeal carcinoma (NPC) is a metastatic Epstein-Barr virus (EBV)-associated cancer that expresses the viral oncogenic protein, latent membrane protein 1 (LMP1). During epithelial metastasis, detached cells must overcome anoikis-induced cell death and gain the ability to reattach and restore growth potential. Anoikis assays have revealed cell survival mechanisms during suspension, but few studies have tracked the fate of cells surviving anoikis-inducing conditions. In this study, a modified anoikis assay was used to examine if the expression of LMP1 confers the recovery of epithelial cells from anoikis. Cells expressing LMP1 mutants and strains were evaluated for distinguishing properties in survival during suspension, reattachment, and outgrowth potential. Expression of LMP1 promoted the outgrowth of the NPC cell line HK1 following anoikis induction that was not attributed to enhanced cell survival in suspension or reattachment. The mechanism of LMP1-induced outgrowth required Akt signaling and the conserved PXQXT motif on LMP1, which activates Akt. Deletion of any of the three LMP1 C-terminal activation regions (CTAR) abrogated anoikis recovery, suggesting that additional LMP1-regulated signaling pathways are likely involved. Of the seven LMP1 strains, only B958, China1, and Med+ promoted HK1 outgrowth from anoikis. This distinguishing biological property segregates LMP1 strains into two categories (anoikis recovering and nonrecovering) and suggests that LMP1 strain-specific sequences may be important in determining metastatic outgrowth potential in NPC tumors.IMPORTANCE LMP1 is one of the most divergent sequences in the EBV genome and phylogenetically segregates into seven LMP1 strains. The LMP1 strains differ in geographical distribution and NPC tumor prevalence, but the molecular basis for this potential selection is not clear. While there are signaling motifs conserved in all LMP1 sequences from circulating EBV isolates, phylogenetic studies of NPC also suggest that there may be sequence selection for tumor-associated LMP1 strains and polymorphisms. The present study describes a modified anoikis assay that can distinguish LMP1 strains into two groups by biological properties. The pleiotropic LMP1 signaling properties and sequence diversity may offer a unique opportunity to illuminate the complex mechanisms of metastasis. Although the host genomic landscape is variable between NPC tumors, the present functional-mapping studies on LMP1 support the notion that viral proteins could serve as molecular tool kits and potentially reveal sequence-associated risk factors in NPC metastatic progression.

New Insights from Elucidating the Role of LMP1 in Nasopharyngeal Carcinoma.

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV) oncogenic protein that has no intrinsic enzymatic activity or sequence homology to cellular or viral proteins. The oncogenic potential of LMP1 has been ascribed to pleiotropic signaling properties initiated through protein-protein interactions in cytosolic membrane compartments, but the effects of LMP1 extend to nuclear and extracellular processes. Although LMP1 is one of the latent genes required for EBV-immortalization of B cells, the biology of LMP1 in the pathogenesis of the epithelial cancer nasopharyngeal carcinoma (NPC) is more complex. NPC is prevalent in specific regions of the world with high incidence in southeast China. The epidemiology and time interval from seroconversion to NPC onset in adults would suggest the involvement of multiple risk factors that complement the establishment of a latent and persistent EBV infection. The contribution of LMP1 to EBV pathogenesis in polarized epithelia has only recently begun to be elucidated. Furthermore, the LMP1 gene has emerged as one of the most divergent sequences in the EBV genome. This review will discuss the significance of recent advances in NPC research from elucidating LMP1 function in epithelial cells and lessons that could be learned from mining LMP1 sequence diversity.

Latent Membrane Protein 1 Is a Novel Determinant of Epstein-Barr Virus Genome Persistence and Reactivation.

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that persistently infects humans, with nearly 95% seropositivity in adults. Infection in differentiating epithelia is permissive, but EBV-associated nasopharyngeal carcinoma (NPC) tumors harbor a clonal and nonproductive latent infection. However, in explanted NPC cultures and epithelial cell lines, episomal EBV genomes are frequently lost. The resulting unstable infection has hampered efforts to study the determinants of EBV persistence and latency in epithelial oncogenesis. The EBV nuclear antigen 1 (EBNA1) protein is required for tethering EBV episomes to cellular DNA and for mitotic segregation to daughter cells. Expression of EBNA1 does not ensure faithful partitioning of EBV episomes or replicons, suggesting that additional regulatory mechanisms have yet to be elucidated. The EBV latent membrane protein 1 (LMP1) is an oncogenic signaling protein expressed in latent and lytic cycles. This study identified that LMP1 contributes to the loss of EBV genomes in latently infected cells and promotes differentiation-induced lytic replication in a polarized air-liquid interface (ALI) culture model. Deletion of LMP1 in recombinantly infected 293 cells promoted the retention of EBV genomes in passaged cells, which was in part localized to a conserved PXQXT motif in the C-terminal signaling domain (CTAR1). Additionally, knockdown of LMP1 in the recombinantly infected NPC cell line HK1 resulted in decreased induction of lytic proteins and infectious EBV titers. These findings are consistent with the hypothesis that in epithelial infections, regulation of LMP1 mechanisms may be a determinant of infection outcome and a potential risk factor for EBV persistence in preneoplastic cells. IMPORTANCE Latent membrane protein 1 (LMP1) is a constitutively active oncogenic signaling protein encoded by Epstein-Barr virus (EBV). Despite monoclonal infection in cases of nasopharyngeal carcinoma (NPC), it has been difficult to reconcile the heterogeneous LMP1 protein levels detected in tumor cells. The LMP1 protein is a pleiotropic signaling protein with oncogenic potential. Findings from this study are consistent with the hypothesis that LMP1 has a role distinct from that of oncogenesis that facilitates the viral life cycle by promoting an unstable but productive infection in differentiating epithelia.

Molecular diversity of IgG responses to Epstein-Barr virus proteins in asymptomatic Epstein-Barr virus carriers.

The Epstein-Barr virus (EBV) is a ubiquitous pathogen that infects over 90 % of adults. EBV is the primary etiological agent of infectious mononucleosis and is closely associated with nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma and Burkitt lymphoma. Clinical serological assays for EBV diagnosis only survey a small portion of the viral proteome, which does not represent the total antigenic breadth presented to the immune system during viral infection. In this study, we have generated an expression library containing the majority of EBV ORFs, and have systematically evaluated IgG responses to those EBV proteins in sera from EBV carriers. In addition to confirming previously recognized dominant EBV antigens, this study has identified additional immunodominant antigens, and has revealed a more expansive antigenic profile of the humoral responses to EBV in asymptomatic carriers. This EBV expression library will be deposited in a public repository with the goal of disseminating this new research tool for the application of identifying potential new biomarkers for EBV-associated diseases.

Regulation of DNA Damage Signaling and Cell Death Responses by Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) and LMP2A in Nasopharyngeal Carcinoma Cells.

UNLABELLED: Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of preneoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins 1 (LMP1) and LMP2A are consistently expressed in NPC and can cooperate in bitransgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were coexpressed in the EBV-negative NPC cell line HK1 and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A coexpression reduced γH2AX (S139) phosphorylation and caspase cleavage induced by a lower dose (5 µM) of the topoisomerase II inhibitor etoposide. Regulation of γH2AX occurred before the onset of caspase activation without modulation of other DNA damage signaling mediators, including ATM, Chk1, or Chk2, and additionally was suppressed by inducers of DNA single-strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A coexpressing cells recovered from cytotoxic doses of etoposide; however, LMP1 expression was sufficient for this effect. LMP1 and LMP2A coexpression did not enhance cell growth, with a moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signaling and cell death activation with no further enhancement in the growth properties of neoplastic cells. IMPORTANCE: NPC is characterized by clonal EBV infection and accounts for >78,000 annual cancer cases with increased incidence in regions where EBV is endemic, such as southeast Asia. The latent proteins LMP1 and LMP2A coexpressed in NPC can individually enhance growth or survival properties in epithelial cells, but their combined effects and potential regulation of DNA repair and checkpoint mechanisms are relatively undetermined. In this study, LMP1-2A coexpression suppressed activation of the DNA damage response (DDR) protein γH2AX induced by selective genotoxins that promote DNA replication stress or SSBs. Expression of LMP1 was sufficient to recover cells, resulting in outgrowth of LMP1 and LMP1-2A-coexpressing cells and indicating distinct LMP1-dependent effects in the restoration of replicative potential. These findings demonstrate novel properties for LMP1 and LMP2A in the cooperative modulation of DDR and apoptotic signaling pathways, further implicating both proteins in the progression of NPC and epithelial malignancies.

Transcriptome changes induced by Epstein-Barr virus LMP1 and LMP2A in transgenic lymphocytes and lymphoma.

Latent membrane protein 1 (LMP1) and LMP2A affect cell growth in both epithelial cells and lymphocytes. In this study, the effects on cellular gene expression were determined by microarray analysis of transgenic mice expressing LMP1, LMP2A, or both using the immunoglobulin heavy chain promoter and enhancer. Large differential changes were detected, indicating that LMP1 and LMP2A can both potently affect host gene transcription, inducing distinct transcriptional profiles. Seventy percent of the changes detected in LMP1/2A doubly transgenic lymphocytes were also modulated by LMP1 or LMP2A alone. These common and unique expression changes indicate that the combined effects of LMP1 and LMP2A may be additive, synergistic, or inhibitory. Using significant pathway analysis, the expression changes detected in LMP1, LMP2A, and LMP1/2A transgenic B lymphocytes were predicted to commonly target cancer and inflammatory pathways. Additionally, using the correlation coefficient to calculate the regulation of known c-Rel and Stat3 transcriptional targets, both were found to be enhanced in LMP1 lymphocytes and lymphomas, and a selection of Stat3 targets was further evaluated and confirmed using quantitative reverse transcription-PCR (RT-PCR). Analyses of the effects on cell growth and viability revealed that LMP2A transgenic lymphocytes had the greatest enhanced viability in vitro; however, doubly transgenic lymphocytes (LMP1/2A) did not have enhanced survival in culture and these mice were similar to negative littermates. These findings indicate that the combined expression of LMP1 and LMP2A has potentially different biological outcomes than when the two proteins are expressed individually. IMPORTANCE The Epstein-Barr virus proteins latent membrane protein 1 (LMP1) and LMP2A have potent effects on cell growth. In transgenic mice that express these proteins in B lymphocytes, the cell growth and survival properties are also affected. LMP1 transgenic mice have increased development of lymphoma, and the LMP1 lymphocytes have increased viability in culture. LMP2A transgenic lymphocytes have altered B cell development and enhanced survival. In this study, analysis of the cellular gene expression changes in transgenic LMP1 and LMP2A lymphocytes and LMP1 lymphomas revealed that both transgenes individually and in combination affected pathways important for the development of cancer and inflammation. Importantly, the combined expression of the two proteins had unique effects on cellular expression and cell viability. This is the first study to look at the combined effects of LMP1 and LMP2A on global changes in host gene expression.

Infection of Epstein-Barr virus in a gastric carcinoma cell line induces anchorage independence and global changes in gene expression.

Latent infection of EBV is linked to the development of multiple cancers that have distinct patterns of expression of viral proteins and microRNAs (miRNAs). In this study, we show that in vitro infection of a gastric epithelial cell line with EBV alters growth properties and induces growth in soft agar. The infected cells have high levels of expression of a large cluster of viral miRNAs, [the BamHI A rightward transcript (BART) miRNAs] and limited viral protein expression. Expression profile microarray analysis of this cell line revealed a large number of changes in cellular expression, with decreased expression of many genes. Inhibition of the trace-expressed levels of the viral oncoprotein, latent membrane protein 1, did not affect growth or alter the pattern of cellular expression. The expression changes are highly enriched for genes involved in cell motility and transformation pathways, suggesting these changes are important for the altered growth phenotype. Importantly, the transcripts decreased by microarray are significantly enriched in both experimentally and bioinformatically predicted BART miRNA targets. The absence of viral protein expression and the enrichment for viral miRNA targets in the modulated cell genes suggest that the BART miRNAs are major contributors to the transformed growth properties of the EBV-infected cells. The ability to affect cell growth through miRNA expression without viral protein expression would be a major factor in the development of cancer in individuals with functional immune systems.

Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) and LMP2A function cooperatively to promote carcinoma development in a mouse carcinogenesis model.

The Epstein-Barr virus (EBV) proteins latent membrane proteins 1 and 2 (LMP1 and LMP2) are frequently expressed in EBV-associated lymphoid and epithelial cancers and have complex effects on cell signaling and growth. The effects of these proteins on epithelial cell growth were assessed in vivo using transgenic mice driven by the keratin 14 promoter (K14). The development of papillomas and carcinomas was determined in the tumor initiator and promoter model using dimethyl benzanthracene (DMBA), followed by repeated treatments of 12-O-tetradecanoyl phorbol 13-acetate (TPA). In these assays, LMP1 functioned as a weak tumor promoter and increased papilloma formation. In contrast, mice expressing LMP2A did not induce or promote papilloma formation. Transgenic LMP1 mice had slightly increased development of squamous cell carcinoma; however, the development of carcinoma was significantly increased in the doubly transgenic mice expressing both LMP1 and LMP2A. DMBA treatment induces an activating mutation in the Harvey-ras (H-ras(61)) oncogene, and this mutation was identified in most papillomas and carcinomas although several papillomas and carcinomas in K14-LMP1 and K14-LMP1/LMP2A mice lacked the mutation. Analysis of signaling pathways that are known to be activated by LMP1 and/or LMP2 indicated that all genotypes had high levels of activated extracellular signal-regulated kinase (ERK) and Stat3 in carcinomas with significantly higher activation in the doubly transgenic carcinomas. These findings suggest that, in combination, LMP1 and LMP2 contribute to carcinoma progression and that this may reflect the combined effects of the proteins on activation of multiple signaling pathways. This study is the first to characterize the effects of LMP2 on tumor initiation and promotion and to identify an effect of the combined expression of LMP1 and LMP2 on the increase of carcinoma development.

Human tumor virus utilizes exosomes for intercellular communication.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is expressed in multiple human malignancies and has potent effects on cell growth. It has been detected in exosomes and shown to inhibit immune function. Exosomes are small secreted cellular vesicles that contain proteins, mRNAs, and microRNAs (miRNAs). When produced by malignant cells, they can promote angiogenesis, cell proliferation, tumor-cell invasion, and immune evasion. In this study, exosomes released from nasopharyngeal carcinoma (NPC) cells harboring latent EBV were shown to contain LMP1, signal transduction molecules, and virus-encoded miRNAs. Exposure to these NPC exosomes activated the ERK and AKT signaling pathways in the recipient cells. Interestingly, NPC exosomes also contained viral miRNAs, several of which were enriched in comparison with their intracellular levels. LMP1 induces expression of the EGF receptor in an EBV-negative epithelial cell line, and exosomes produced by these cells also contain high levels of EGF receptor in exosomes. These findings suggest that the effects of EBV and LMP1 on cellular expression also modulate exosome content and properties. The exosomes may manipulate the tumor microenvironment to influence the growth of neighboring cells through the intercellular transfer of LMP1, signaling molecules, and viral miRNAs.

Epstein-Barr virus latent membrane protein-1 effects on junctional plakoglobin and induction of a cadherin switch.

Latent membrane protein-1 (LMP1) is considered the major oncoprotein of Epstein-Barr virus and is frequently expressed in nasopharyngeal carcinoma (NPC). LMP1 promotes growth and migration of epithelial cells, and the loss of plakoglobin has been identified as a contributing factor to LMP1-induced migration. Plakoglobin is a junctional protein that can also serve as a transcription factor in Tcf/Lef signaling. To determine the effects of LMP1 on the molecular and functional properties of plakoglobin, LMP1 was overexpressed in the NPC cell line C666-1. LMP1 did not affect plakoglobin stability but did decrease plakoglobin transcription. The resultant decreased levels of nuclear plakoglobin did not affect Tcf/Lef activity or the amount of plakoglobin bound to Tcf4. Although LMP1 induced and stabilized beta-catenin, a protein with common binding partners to plakoglobin, the loss of plakoglobin did not affect its association with Tcf4. However, LMP1 did induce a cadherin switch from E- to N-cadherin, a process involved in cancer progression, and enhanced the association of junctional beta-catenin with N-cadherin. LMP1 decreased overall levels of junctional plakoglobin but the remaining junctional plakoglobin was found associated with the induced N-cadherin. This increased association of junctional plakoglobin with N-cadherin was a distinguishing feature of LMP1-expressing cells that have reduced migration due to restoration of plakoglobin. Low levels of plakoglobin were also detected in human NPC tissues. These findings reveal that the effects of LMP1 on junctional plakoglobin and the initiation of a cadherin switch likely contribute to metastasis of NPC.

EBV latent membrane protein 1 effects on plakoglobin, cell growth, and migration.

Latent membrane protein 1 (LMP1), the major oncoprotein of EBV, is likely responsible for many of the altered cellular growth properties in EBV-associated cancers, including nasopharyngeal carcinoma (NPC). In this study, the effects of LMP1 on cell growth and migration were studied in the context of the EBV-positive C666-1 NPC cell line. In the soft agar transformation and Transwell metastasis assays, LMP1 enhanced cell growth and migration through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappaB (NF-kappaB) signaling. Inhibitors of PI3K, Akt, and NF-kappaB signaling dramatically reduced these enhanced properties. An IkappaBalpha super-repressor also blocked these effects. However, constitutive activation of Akt alone did not alter cell growth, suggesting that both PI3K/Akt and NF-kappaB activation are required by LMP1. These enhanced effects required the full-length LMP1 encompassing both the PI3K/Akt-activating COOH-terminal activation region (CTAR) 1 and the nonredundant NF-kappaB-activating regions CTAR1 and CTAR2. LMP2A, a latent protein that is also frequently expressed in NPC, similarly activates the PI3K/Akt pathway; however, its overexpression in C666-1 cells did not affect cell growth or migration. LMP1 also decreased expression of the junctional protein plakoglobin, which was shown to be partially responsible for enhanced migration induced by LMP1. This study reveals that in epithelial cells the transforming properties of LMP1 require activation of both PI3K/Akt and NF-kappaB and shows that the loss of plakoglobin expression by LMP1 is a significant factor in the enhanced migration.

EBV latent membrane protein 1 activates Akt, NFkappaB, and Stat3 in B cell lymphomas.

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.