EBV-encoded Nuclear Antigen Research Articles

Epstein-Barr virus–encoded EBNA2 downregulates ICOSL by inducing miR-24 in B-cell lymphoma

AbstractHematological malignancies such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B-cell lymphoma (DLBCL) cause significant morbidity in humans. A substantial number of these lymphomas, particularly HL and DLBCLs have poorer prognosis because of their association with Epstein-Barr virus (EBV). Our earlier studies have shown that EBV-encoded nuclear antigen (EBNA2) upregulates programmed cell death ligand 1 in DLBCL and BLs by downregulating microRNA-34a. Here, we investigated whether EBNA2 affects the inducible costimulator (ICOS) ligand (ICOSL), a molecule required for efficient recognition of tumor cells by T cells through the engagement of ICOS on the latter. In virus-infected and EBNA2-transfected B-lymphoma cells, ICOSL expression was reduced. Our investigation of the molecular mechanisms revealed that this was due to an increase in microRNA-24 (miR-24) by EBNA2. By using ICOSL 3′ untranslated region–luciferase reporter system, we validated that ICOSL is an authentic miR-24 target. Transfection of anti–miR-24 molecules in EBNA2-expressing lymphoma cells reconstituted ICOSL expression and increased tumor immunogenicity in mixed lymphocyte reactions. Because miR-24 is known to target c-MYC, an oncoprotein positively regulated by EBNA2, we analyzed its expression in anti–miR-24 transfected lymphoma cells. Indeed, the reduction of miR-24 in EBNA2-expressing DLBCL further elevated c-MYC and increased apoptosis. Consistent with the in vitro data, EBNA2-positive DLBCL biopsies expressed low ICOSL and high miR-24. We suggest that EBV evades host immune responses through EBNA2 by inducing miR-24 to reduce ICOSL expression, and for simultaneous rheostatic maintenance of proproliferative c-MYC levels. Overall, these data identify miR-24 as a potential therapeutically relevant target in EBV-associated lymphomas.

Epstein-Barr Virus Synergizes with BRD7 to Conquer c-Myc-Mediated Viral Latency Maintenance via Chromatin Remodeling.

Epstein-Barr virus (EBV) switches between latent and lytic phases in hosts, which is important in the development of related diseases. However, the underlying mechanism of controlling the viral biphasic life cycle and how EBV mediates this regulation remain largely unknown. This study identified bromodomain-containing protein 7 (BRD7) as a crucial host protein in EBV latent infection. Based on the chromatin immunoprecipitation (ChIP) sequencing of endogenous BRD7 in Burkitt lymphoma cells, we found that EBV drove BRD7 to regulate cellular and viral genomic loci, including the transcriptional activation of c-Myc, a recently reported regulator of EBV latency. Additionally, EBV-mediated BRD7 signals were enriched around the FUSE (far-upstream sequence element) site in chromosome 8 and the enhancer LOC108348026 in the lgH locus, which might activate the c-Myc alleles. Mechanically, EBV-encoded nuclear antigen 1 (EBNA1) bound to BRD7 and colocalized at promoter regions of the related genes, thus serving as cofactors for the maintenance of viral latency. Moreover, the disruption of BRD7 decreased the c-Myc expression, induced the BZLF1 expression, and reactivated the lytic cycle. Our findings reveal the unique role of BRD7 to synergize with EBV in maintaining the viral latency state via chromatin remodeling. This study paves the way for understanding the new molecular mechanism of EBV-induced chromatin remodeling and latent-lytic switch, providing novel therapeutic candidate targets for EBV persistent infection. IMPORTANCE When establishing persistent infection in most human hosts, EBV is usually latent. How the viral latency is maintained in cells remains largely unknown. c-Myc was recently reported to act as a controller of the lytic switch, while whether and how EBV regulates it remain to be explored. Here, we identified that BRD7 is involved in controlling EBV latency. We found that EBV-mediated BRD7 was enriched in both the normal promoter regions and the translocation alleles of c-Myc, and disruption of BRD7 decreased c-Myc expression to reactivate the lytic cycle. We also demonstrated that EBV-encoded EBNA1 bound to and regulated BRD7. Therefore, we reveal a novel mechanism by which EBV can regulate its infection state by coordinating with host BRD7 to target c-Myc. Our findings will help future therapeutic intervention strategies for EBV infection and pathogenesis.

Establishment and characterization of two Epstein-Barr virus-positive gastric cancer cell lines with epitheliotropic M81 strain undergoing distinct viral and altered cellular expression profiles.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer (GC) distinguished by the presence of the EBV genome and limited viral gene expression within malignant epithelial cells. EBV infection is generally thought to be a relatively late event following atrophic gastritis in carcinogenesis, which implies the heterogeneity of EBVaGC. To facilitate the study of the role of EBV in EBVaGC, we established two EBV-positive GC cell lines (AGS-EBV and HGC27-EBV) with an epitheliotropic EBV strain M81 and characterized viral and cellular gene expression profiles in comparison to SNU719, a naturally derived EBV-positive GC cell line. Like SNU719, AGS-EBV and HGC27-EBV stably maintained their EBV genomes and expressed EBV-encoded small RNAs and nuclear antigen EBNA1. Comprehensive analysis of the expression of EBV-encoded miRNAs within the BamHI-A region rightward transcript region, and the transcripts of EBV latent and lytic genes in cell lines, as well as xenografts, reveals that AGS-EBV and HGC27-EBV cells undergo distinct viral expression profiles. A very small fraction of AGS-EBV and SNU719 cells can spontaneously produce infectious progeny virions, while HGC27-EBV does not. AGS-EBV (both M81 and Akata) cells largely mimic SNU719 cells in viral gene expression profiles, and altered cellular functions and pathways perturbed by EBV infection. Phylogenetic analysis of the EBV genome shows both M81 and Akata EBV strains are closely related to clinical EBVaGC isolates. Taken together, these two newly established EBV-positive GC cell lines can serve as models to further investigate the role of EBV in different contexts of gastric carcinogenesis and identify novel therapeutics against EBVaGC.

Saliva antibody-fingerprint of reactivated latent viruses after mild/asymptomatic COVID-19 is unique in patients with myalgic-encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic disease considered to be triggered by viral infections in a majority of cases. Symptoms overlap largely with those of post-acute sequelae of COVID-19/long-COVID implying common pathogenetic mechanisms. SARS-CoV-2 infection is risk factor for sustained latent virus reactivation that may account for the symptoms of post-viral fatigue syndromes. The aim of this study was first to investigate whether patients with ME/CFS and healthy donors (HDs) differed in their antibody response to mild/asymptomatic SARS-CoV-2 infection. Secondly, to analyze whether COVID-19 imposes latent virus reactivation in the cohorts. Anti-SARS-CoV-2 antibodies were analyzed in plasma and saliva from non-vaccinated ME/CFS (n=95) and HDs (n=110) using soluble multiplex immunoassay. Reactivation of human herpesviruses 1-6 (HSV1, HSV2, VZV, EBV, CMV, HHV6), and human endogenous retrovirus K (HERV-K) was detected by anti-viral antibody fingerprints in saliva. At 3-6 months after mild/asymptomatic SARS-CoV-2 infection, virus-specific antibodies in saliva were substantially induced signifying a strong reactivation of latent viruses (EBV, HHV6 and HERV-K) in both cohorts. In patients with ME/CFS, antibody responses were significantly stronger, in particular EBV-encoded nuclear antigen-1 (EBNA1) IgG were elevated in patients with ME/CFS, but not in HDs. EBV-VCA IgG was also elevated at baseline prior to SARS-infection in patients compared to HDs. Our results denote an altered and chronically aroused anti-viral profile against latent viruses in ME/CFS. SARS-CoV-2 infection even in its mild/asymptomatic form is a potent trigger for reactivation of latent herpesviruses (EBV, HHV6) and endogenous retroviruses (HERV-K), as detected by antibody fingerprints locally in the oral mucosa (saliva samples). This has not been shown before because the antibody elevation is not detected systemically in the circulation/plasma.

Epstein-Barr virus infection patterns in nodular lymphocyte-predominant Hodgkin lymphoma.

To investigate Epstein-Barr virus (EBV) latency types in 19 cases of EBV-positive nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), as such information is currently incomplete. Immunohistochemistry (IHC) for CD20, CD79a, PAX5, OCT2, CD30, CD15, CD3 and programmed cell death protein 1 was performed. For EBV detection, in-situ hybridisation (ISH) for EBV-encoded RNA (EBER) was employed combined with IHC for EBV-encoded latent membrane protein (LMP)-1, EBV-encoded nuclear antigen (EBNA)-2, and EBV-encoded BZLF1. In 95% of the cases, neoplastic cells with features of Hodgkin and Reed-Sternberg (HRS) cells were present, mostly showing expression of CD30. In all cases, the B-cell phenotype was largely intact, and delineation from classic Hodgkin lymphoma (CHL) was further supported by myocyte enhancer factor 2B (MEF2B) detection. All tumour cells were EBER-positive except in two cases. EBV latency type II was most frequent (89%) and type I was rare. Cases with latency type I were CD30-negative. Five cases contained some BZLF1-positive and/or EBNA-2-positive bystander lymphocytes. As HRS morphology of neoplastic cells and CD30 expression are frequent features of EBV-positive NLPHL, preservation of the B-cell transcription programme, MEF2B expression combined with NLPHL-typical architecture and background composition facilitate distinction from CHL. EBER ISH is the method of choice to identify these cases. The majority present with EBV latency type II, and only rare cases present with latency type I, which can be associated with missing CD30 expression. The presence of occasional bystander lymphocytes expressing BZLF1 and/or EBNA-2 and the partial EBV infection of neoplastic cells in some cases could indicate that EBV is either not primarily involved or is only a transient driver in the pathogenesis of EBV-positive NLPHL.

'Off-the-shelf’ allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies

BackgroundEpstein-Barr virus (EBV), an oncogenic human gammaherpesvirus, is associated with a wide range of human malignancies of epithelial and B-cell origin. Recent studies have demonstrated promising safety and clinical efficacy...

EBV-EBNA1 constructs an immunosuppressive microenvironment for nasopharyngeal carcinoma by promoting the chemoattraction of Treg cells

BackgroundNasopharyngeal carcinoma (NPC) is primarily caused by the Epstein-Barr virus (EBV) infection in NPC endemic areas. EBNA1 is an EBV-encoded nuclear antigen, which plays a critical role in the maintenance...

The Epstein-Barr Virus-Encoded EBNA1 Protein Activates the Bone Morphogenic Protein (BMP) Signalling Pathway to Promote Carcinoma Cell Migration.

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) protein is expressed in all virus-associated malignancies, where it performs an essential role in the maintenance, replication and transcription of the EBV genome. In recent years, it has become apparent that EBNA1 can also influence cellular gene transcription. Here, we demonstrate that EBNA1 is able to stimulate the expression of the Transforming growth factor-beta (TGFβ) superfamily member, bone morphogenic protein 2 (BMP2), with consequential activation of the BMP signalling pathway in carcinoma cell lines. We show that BMP pathway activation is associated with an increase in the migratory capacity of carcinoma cells, an effect that can be ablated by the BMP antagonist, Noggin. Gene expression profiling of authentic EBV-positive nasopharyngeal carcinoma (NPC) tumours revealed the consistent presence of BMP ligands, established BMP pathway effectors and putative target genes, constituting a prominent BMP “signature” in this virus-associated cancer. Our findings show that EBNA1 is the major viral-encoded protein responsible for activating the BMP signalling pathway in carcinoma cells and supports a role for this pathway in promoting cell migration and possibly, metastatic spread.

Conditional expression of a dominant-negative form of Epstein-Barr virus (EBV) nuclear antigen EBNALP inhibits EBV-positive lymphoblastoid cell growth.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that transforms primary B lymphocytes, yielding lymphoblastoid cell lines (LCLs). EBV-encoded nuclear antigen 2 (EBNA2) and EBV-encoded nuclear antigen leader protein (EBNALP) are the first viral products expressed after EBV infection of primary B lymphocytes and are essential for EBV-induced B-lymphocyte growth transformation. EBNA2 functions as a transcriptional activator of viral and cellular genes, with EBNALP as a coactivator for EBNA2-mediated transcriptional activation. We previously reported that mutant EBNALP with a C-terminal 10-amino-acid truncation loses the ability to coactivate, and has a dominant-negative effect on wild-type-EBNALP-mediated coactivation. However, the functional relevance of EBNALP in maintenance of LCL cell growth has not been investigated. To address this, we have constructed LCL-derived cell clones in which this dominant-negative form of EBNALP (DNLP) is conditionally expressed by the Cre-loxP system. We used these cells to evaluate the effect of DNLP expression on EBV-induced cell proliferation. After drug treatment, the DNLP-expressing LCL clones showed reduced cell proliferation and viability. These results indicate that EBNALP is critical for maintaining LCL growth and EBV-induced cell proliferation.

EBNA1: Oncogenic Activity, Immune Evasion and Biochemical Functions Provide Targets for Novel Therapeutic Strategies against Epstein-Barr Virus- Associated Cancers.

The presence of the Epstein-Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) protein in all EBV-carrying tumours constitutes a marker that distinguishes the virus-associated cancer cells from normal cells and thereby offers opportunities for targeted therapeutic intervention. EBNA1 is essential for viral genome maintenance and also for controlling viral gene expression and without EBNA1, the virus cannot persist. EBNA1 itself has been linked to cell transformation but the underlying mechanism of its oncogenic activity has been unclear. However, recent data are starting to shed light on its growth-promoting pathways, suggesting that targeting EBNA1 can have a direct growth suppressing effect. In order to carry out its tasks, EBNA1 interacts with cellular factors and these interactions are potential therapeutic targets, where the aim would be to cripple the virus and thereby rid the tumour cells of any oncogenic activity related to the virus. Another strategy to target EBNA1 is to interfere with its expression. Controlling the rate of EBNA1 synthesis is critical for the virus to maintain a sufficient level to support viral functions, while at the same time, restricting expression is equally important to prevent the immune system from detecting and destroying EBNA1-positive cells. To achieve this balance EBNA1 has evolved a unique repeat sequence of glycines and alanines that controls its own rate of mRNA translation. As the underlying molecular mechanisms for how this repeat suppresses its own rate of synthesis in cis are starting to be better understood, new therapeutic strategies are emerging that aim to modulate the translation of the EBNA1 mRNA. If translation is induced, it could increase the amount of EBNA1-derived antigenic peptides that are presented to the major histocompatibility (MHC) class I pathway and thus, make EBV-carrying cancers better targets for the immune system. If translation is further suppressed, this would provide another means to cripple the virus.

Latent Membrane Protein 1 (LMP1) and LMP2A Collaborate To Promote Epstein-Barr Virus-Induced B Cell Lymphomas in a Cord Blood-Humanized Mouse Model but Are Not Essential.

Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The ability of EBV to convert human B cells into long-lived lymphoblastoid cell lines (LCLs) in vitro requires the collaborative effects of EBNA2 (which hijacks Notch signaling), latent membrane protein 1 (LMP1) (which mimics CD40 signaling), and EBV-encoded nuclear antigen 3A (EBNA3A) and EBNA3C (which inhibit oncogene-induced senescence and apoptosis). However, we recently showed that an LMP1-deleted EBV mutant induces B cell lymphomas in a newly developed cord blood-humanized mouse model that allows EBV-infected B cells to interact with CD4 T cells (the major source of CD40 ligand). Here we examined whether the EBV LMP2A protein, which mimics constitutively active B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A delays the onset of EBV-induced lymphomas but does not affect the tumor phenotype or the number of tumors. The simultaneous deletion of both LMP1 and LMP2A results in fewer tumors and a further delay in tumor onset. Nevertheless, the LMP1/LMP2A double mutant induces lymphomas in approximately half of the infected animals. These results indicate that neither LMP1 nor LMP2A is absolutely essential for the ability of EBV to induce B cell lymphomas in the cord blood-humanized mouse model, although the simultaneous loss of both LMP1 and LMP2A decreases the proportion of animals developing tumors and increases the time to tumor onset. Thus, the expression of either LMP1 or LMP2A may be sufficient to promote early-onset EBV-induced tumors in this model.IMPORTANCE EBV causes human lymphomas, but few models are available for dissecting how EBV causes lymphomas in vivo in the context of a host immune response. We recently used a newly developed cord blood-humanized mouse model to show that EBV can cooperate with human CD4 T cells to cause B cell lymphomas even when a major viral transforming protein, LMP1, is deleted. Here we examined whether the EBV protein LMP2A, which mimics B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A alone has little effect on the ability of EBV to cause lymphomas but delays tumor onset. The deletion of both LMP1 and LMP2A results in a smaller number of lymphomas in infected animals, with an even more delayed time to tumor onset. These results suggest that LMP1 and LMP2A collaborate to promote early-onset lymphomas in this model, but neither protein is absolutely essential.

Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr Virus episomal latency.

Epstein-Barr Virus (EBV) latent infection is a causative co-factor for endemic Nasopharyngeal Carcinoma (NPC). NPC-associated variants have been identified in EBV-encoded nuclear antigen EBNA1. Here, we solve the X-ray crystal structure of an NPC-derived EBNA1 DNA binding domain (DBD) and show that variant amino acids are found on the surface away from the DNA binding interface. We show that NPC-derived EBNA1 is compromised for DNA replication and episome maintenance functions. Recombinant virus containing the NPC EBNA1 DBD are impaired in their ability to immortalize primary B-lymphocytes and suppress lytic transcription during early stages of B-cell infection. We identify Survivin as a host protein deficiently bound by the NPC variant of EBNA1 and show that Survivin depletion compromises EBV episome maintenance in multiple cell types. We propose that endemic variants of EBNA1 play a significant role in EBV-driven carcinogenesis by altering key regulatory interactions that destabilize latent infection.

Epstein-Barr virus and carcinomas: rare association of the virus with gastric adenocarcinomas.

We have analysed 174 gastric carcinomas from the United Kingdom and from Japan for the presence of Epstein-Barr virus (EBV) using in situ hybridisation for the small EBV-encoded nuclear RNAs (EBERs). EBV was detected in the tumour cells in all of six undifferentiated gastric carcinomas with prominent lymphoid stroma (undifferentiated carcinomas of nasopharyngeal type, UCNT) but only in three of the remaining 168 typical gastric adenocarcinomas (1.8%). No differences were observed between the British and the Japanese cases. One case with an EBV-positive UCNT showed adjacent areas of EBV-negative typical adenocarcinoma. It is uncertain whether these patterns represent two independent carcinomas or whether they are the result of heterogeneous EBV infection in a single tumour. In the remaining EBV-positive carcinomas, viral transcripts were detected in virtually all tumour cells, indicating that EBV infection must have taken place early in the neoplastic process and suggesting that the virus is likely to be of pathogenetic significance for the virus-associated tumours. Immunohistology demonstrated absence of detectable levels of the EBV-encoded latent membrane protein, LMP1, and nuclear antigen, EBNA2. The BZLF1 protein which induces the switch from latent to lytic infection was demonstrated in a small proportion of the tumour cells in three cases. The close association of EBV with undifferentiated gastric carcinomas compared to the variable association with gastric adenocarcinomas suggests fundamentally different roles for the virus in the aetiology of these two malignancies.

ERK2 phosphorylation of EBNA1 serine 383 residue is important for EBNA1-dependent transactivation.

Functional inhibition of Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) can cause the death of EBV infected cells. In this study, a bioinformatics tool predicted the existence of putative extracellular signal-regulated kinase (ERK) docking and substrate consensus sites on EBNA1, suggesting that ERK2 could bind to and phosphorylate EBNA1. In accordance, ERK2 was found to phosphorylate EBNA1 serine 383 in a reaction suppressed by H20 (a structural congener of the ERK inhibitor), U0126 (an inhibitor of MEK kinase), and mutations at substrate (S383A) or putative ERK docking sites. Wild-type (S383) and phosphomimetic (S383D) EBNA1 demonstrated comparable transactivation function, which was suppressed by H20 or U0126. In contrast, non-phosphorylated EBNA1 mutants displayed significantly impaired transactivation activity. ERK2 knock-down by siRNA, or treatment with U0126 or H20 repressed EBNA1-dependent transactivation. Collectively, these data indicate that blocking ERK2-directed phosphorylation can suppress EBNA1-transactivation function in latent EBV-infected cells, validating ERK2 as a drug target for EBV-associated disorders.

Identification of MEF2B , EBF1 , and IL6R as Direct Gene Targets of Epstein-Barr Virus (EBV) Nuclear Antigen 1 Critical for EBV-Infected B-Lymphocyte Survival

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the EBV-encoded nuclear antigen and sequence-specific DNA binding protein required for viral origin binding and episome maintenance during latency. EBNA1 can also bind to numerous sites in the cellular genome and can provide a host cell survival function, but it is not yet known how EBNA1 sequence-specific binding is responsible for host cell survival. Here, we integrate EBNA1 chromatin immunoprecipitation sequencing (ChIP-Seq) with transcriptome sequencing (RNA-Seq) after EBNA1 depletion to identify cellular genes directly regulated by EBNA1 that are also essential for B-cell survival. We first compared EBNA1 ChIP-Seq patterns in four different EBV-positive cell types, including Burkitt lymphoma (BL) cells, nasopharyngeal carcinoma (NPC) cells, and lymphoblastoid cell lines (LCLs). EBNA1 binds to ~1,000 sites that are mostly invariant among cell types and share a consensus recognition motif. We found that a large subset of EBNA1 binding sites are located proximal to transcription start sites and correlate genome-wide with transcription activity. EBNA1 bound to genes of high significance for B-cell growth and function, including MEF2B, IL6R, and EBF1. EBNA1 depletion from latently infected LCLs results in the loss of cell proliferation and the loss of gene expression for some EBNA1-bound genes, including MEF2B, EBF1, and IL6R. Depletion of MEF2B, EBF1, or IL6R partially phenocopies EBNA1 depletion by decreasing the cell growth and viability of cells latently infected with EBV. These findings suggest that EBNA1 binds to a large cohort of cellular genes important for cell viability and implicates EBNA1 as a critical regulator of transcription of host cell genes important for enhanced survival of latently infected cells. Epstein-Barr virus (EBV) latent infection is responsible for a variety of lymphoid and epithelial cell malignancies. EBNA1 is the EBV-encoded nuclear antigen that is consistently expressed in all EBV-associated cancers. EBNA1 is known to provide a host cell survival function, but the mechanism is not known. EBNA1 is a sequence-specific binding protein important for viral genome maintenance during latency. Here, by integrating ChIP-Seq and RNA-Seq, we demonstrate that EBNA1 binds directly to the promoter regulatory regions and upregulates the transcription of host genes that are important for the survival of EBV-infected cells. Identification of EBNA1 target genes provides potential new targets for therapeutic intervention in EBV-associated disease.

Epstein-Barr virus latent genes.

Latent Epstein–Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.

MRNA Structural constraints on EBNA1 synthesis impact on in vivo antigen presentation and early priming of CD8+ T cells.

Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8+ T cell epitopes by CD11c+ dendritic cells in draining lymph nodes and early priming of antigen-specific CD8+ T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8+ T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection.

Characterization of Spontaneous Malignant Lymphomas in Japanese Macaques (Macaca fuscata)

Lymphomas are common spontaneous tumors in nonhuman primates but remain poorly characterized in Japanese macaques (Macaca fuscata). This study examined 5 cases of spontaneous malignant lymphoma in Japanese macaques, focusing on the immunophenotypes and presence of simian lymphocryptoviruses, which are Epstein-Barr virus-related herpesviruses in nonhuman primates. The macaques with lymphoma were 5 to 28 years old, indicating that lymphomas develop over a wide age range. The common macroscopic findings were splenomegaly and enlargement of lymph nodes. Histologic and immunohistochemical analyses revealed that all cases were non-Hodgkin type and exhibited a T-cell phenotype, positive for CD3 but negative for CD20 and CD79α. The lymphomas exhibited diverse cellular morphologies and were subdivided into 3 types according to the World Health Organization classification. These included 3 cases of peripheral T-cell lymphoma, not otherwise specified; 1 case of T-cell prolymphocytic leukemia; and 1 case of an unclassifiable T-cell lymphoma. Positive signals were detected by in situ hybridization in 2 of the 4 examined cases using probes for the Epstein-Barr virus-encoded small RNA (EBER). Furthermore, the presence of M. fuscata lymphocryptovirus 2, a macaque homolog of Epstein-Barr virus, was demonstrated in EBER-positive cases by polymerase chain reaction amplification followed by direct sequencing. Immunohistochemistry using antibody to the Epstein-Barr virus-encoded nuclear antigen 2 was negative, even in the EBER-positive cases. The present study suggests that T-cell lymphoma is more common than B-cell lymphoma in Japanese macaques and that M. fuscata lymphocryptovirus 2 is present in some cases.

G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation.

Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs.

Small molecule inhibition of Epstein–Barr virus nuclear antigen-1 DNA binding activity interferes with replication and persistence of the viral genome

The replication and persistence of extra chromosomal Epstein–Barr virus (EBV) episome in latently infected cells are primarily dependent on the binding of EBV-encoded nuclear antigen 1 (EBNA1) to the cognate EBV oriP element. In continuation of the previous study, herein we characterized EBNA1 small molecule inhibitors (H20, H31) and their underlying inhibitory mechanisms. In silico docking analyses predicted that H20 fits into a pocket in the EBNA1 DNA binding domain (DBD). However, H20 did not significantly affect EBNA1 binding to its cognate sequence. A limited structure-relationship study of H20 identified a hydrophobic compound H31, as an EBNA1 inhibitor. An in vitro EBNA1 EMSA and in vivo EGFP-EBNA1 confocal microscopy analysis showed that H31 inhibited EBNA1-dependent oriP sequence-specific DNA binding activity, but not sequence-nonspecific chromosomal association. Consistent with this, H31 repressed the EBNA1-dependent transcription, replication, and persistence of an EBV oriP plasmid. Furthermore, H31 induced progressive loss of EBV episome. In addition, H31 selectively retarded the growth of EBV-infected LCL or Burkitt’s lymphoma cells. These data indicate that H31 inhibition of EBNA1-dependent DNA binding decreases transcription from and persistence of EBV episome in EBV-infected cells. These new compounds might be useful probes for dissecting EBNA1 functions in vitro and in vivo.