Genetic variability and mutation of Epstein‒Barr virus (EBV)-encoded LMP-1 and BHRF-1 genes in EBV-infected patients: identification of precise targets for development of personalized EBV vaccines.

The critical Epstein‒Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) and BamHI fragment H rightward open reading frame 1 (BHRF-1) genes affect EBV-mediated malignant transformation and virus replication during EBV infection. Therefore, these two genes are considered ideal targets for EBV vaccine development. However, gene mutations in LMP-1 and BHRF-1 in different cohorts may affect the biological functions of EBV, which would seriously hinder development of personalized vaccines for EBV. In the present study, by performing nested polymerase chain reaction (nested PCR) and DNA sequence techniques, we analyzed the nucleotide variability and phylogeny of LMP-1 containing a 30 bp deletion region (del-LMP-1) and BHRF-1 in EBV-infected patients (N = 382) and healthy persons receiving physical examination (N = 98; defined as the control group) in Yunnan Province, China. Three BHRF-1 subtypes were identified in this study: 79V88V, 79L88L, and 79V88L, with mutation frequencies of 58.59%, 24.24%, and 17.17%, respectively. Compared with the control group, the distribution of BHRF-1 subtypes of the three groups showed no significant difference, suggesting that BHRF-1 is highly conserved in EBV-related samples. In addition, a short fragment of del-LMP-1 was found in 133 cases, and the nucleotide variation rate was 87.50% (133/152). For del-LMP-1, a significant distribution in three groups was detected, as characterized by a high mutation rate. In conclusion, our study illustrates gene variability and mutations of EBV-encoded del-LMP-1 and BHRF-1 in clinical samples. Highly mutated LMP-1 might be associated with various types of EBV-related diseases, indicating that BHRF-1 combined with LMP-1 may be used as an ideal target for development of EBV personalized vaccines.

Crystal Structures of Epstein-Barr Virus Bcl-2 Homolog BHRF1 Bound to Bid and Puma BH3 Motif Peptides.

Apoptosis is a powerful defense mechanism used by multicellular organisms to counteract viral infection. In response to premature host cell suicide, viruses have evolved numerous countermeasures to ensure cell viability to optimize their replication by encoding proteins homologous in structure and function to cellular pro-survival Bcl-2 proteins. Epstein-Barr virus (EBV), a member of the Gammaherpesviridae, encodes the Bcl-2 homolog BHRF1, a potent inhibitor of Bcl-2-mediated apoptosis. BHRF1 acts by directly targeting Bid and Puma, two proapoptotic proteins of the Bcl-2 family. Here, we determined the crystal structures of BHRF1 bound to peptides spanning the Bcl-2 binding motifs (Bcl-2 homology 3 motif, BH3) of Bid and Puma. BHRF1 engages BH3 peptides using the canonical ligand-binding groove of its Bcl-2 fold and maintains a salt bridge between an Arg residue with a conserved Asp residue in the BH3 motif mimicking the canonical ionic interaction seen in host Bcl-2:BH3 motif complexes. Furthermore, both Bid and Puma utilize a fifth binding pocket in the canonical ligand binding groove of BHRF1 to provide an additional hydrophobic interaction distinct from the interactions previously seen with Bak and Bim. These findings provide a structural basis for EBV-mediated suppression of host cell apoptosis and reveal the flexibility of virus encoded Bcl-2 proteins in mimicking key interactions from the endogenous host signaling pathways.

Virus-Mediated Inhibition of Apoptosis in the Context of EBV-Associated Diseases: Molecular Mechanisms and Therapeutic Perspectives.

Epstein-Barr virus (EBV), the representative of the Herpesviridae family, is a pathogen extensively distributed in the human population. One of its most characteristic features is the capability to establish latent infection in the host. The infected cells serve as a sanctuary for the dormant virus, and therefore their desensitization to apoptotic stimuli is part of the viral strategy for long-term survival. For this reason, EBV encodes a set of anti-apoptotic products. They may increase the viability of infected cells and enhance their resistance to chemotherapy, thereby contributing to the development of EBV-associated diseases, including Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), gastric cancer (GC), nasopharyngeal carcinoma (NPC) and several other malignancies. In this paper, we have described the molecular mechanism of anti-apoptotic actions of a set of EBV proteins. Moreover, we have reviewed the pro-survival role of non-coding viral transcripts: EBV-encoded small RNAs (EBERs) and microRNAs (miRNAs), in EBV-carrying malignant cells. The influence of EBV on the expression, activity and/or intracellular distribution of B-cell lymphoma 2 (Bcl-2) protein family members, has been presented. Finally, we have also discussed therapeutic perspectives of targeting viral anti-apoptotic products or their molecular partners.

Role of Epstein-Barr Virus C Promoter Deletion in Diffuse Large B Cell Lymphoma.

The Epstein-Barr virus (EBV) is the cause of several malignancies, including diffuse large B cell lymphoma (DLBCL). We recently found that EBV genomes in EBV-positive cancer specimens have various deletions (Okuno et al. Nat Microbiol. 2019). Here, we focus on the deletion of C promoter (Cp), which transcribes EBV nuclear antigen (EBNA) genes in type III latency. The Cp deletion found in a DLBCL patient (332 bp) was introduced into EBV-BAC of the B95-8 strain. Interestingly, the dCp virus transformed B cells more efficiently than WT and revertant strains. Deletion of Cp also promoted tumor formation and severe pathogenicity in a mouse xenograft model. RNA sequencing and qRT-PCR analyses revealed that Cp transcription was undetectable in the dCp cells. Instead, transcription from the W promoter (Wp), an alternative promoter for EBNA, was activated in the dCp mutant. We also found that the expression of latent membrane protein 2A (LMP2A) was somehow induced in the dCp mutant. Double knockout of Cp and LMP2A indicated that LMP2A is crucial for B cell transformation, but the increased transformation induced by Cp deletion cannot be explained by LMP2A alone. We also tested the effect of an anti-apoptotic viral BCL2 homolog, BHRF1, because its expression was reportedly induced more efficiently by that of Wp. However, increased growth transformation via Cp deletion was not due to the BHRF1 gene. Taken together, the results indicated that deletion of a specific region in Cp increased in vitro transformation and the rate of progression of EBV-positive lymphoproliferative disorders in vivo. Our data suggest that genomic alteration not only of the host but also the virus promotes EBV-positive tumor generation and expansion, although the molecular mechanism underlying this phenomenon is still unclear. However, LMP2A and BHRF1 are not involved.

RNA m6 A methylation regulates virus-host interaction and EBNA2 expression during Epstein-Barr virus infection.

INTRODUCTION: N6 -methyladenosine (m6 A) is the most prevalent modification that occurs in messenger RNA (mRNA), affecting mRNA splicing, translation, and stability. This modification is reversible, and its related biological functions are mediated by "writers," "erasers," and "readers." The field of viral epitranscriptomics and the role of m6 A modification in virus-host interaction have attracted much attention recently. When Epstein-Barr virus (EBV) infects a human B lymphocyte, it goes through three phases: the pre-latent phase, latent phase, and lytic phase. Little is known about the viral and cellular m6 A epitranscriptomes in EBV infection, especially in the pre-latent phase during de novo infection. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-RT-qPCR were used to determine the m6 A-modified transcripts during de novo EBV infection. RIP assay was used to confirm the binding of EBNA2 and m6 A readers. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to test the effect of m6 A on the host and viral gene expression. RESULTS: Here, we provided mechanistic insights by examining the viral and cellular m6 A epitranscriptomes during de novo EBV infection, which is in the pre-latent phase. EBV EBNA2 and BHRF1 were highly m6 A-modified upon EBV infection. Knockdown of METTL3 (a "writer") decreased EBNA2 expression levels. The emergent m6 A modifications induced by EBV infection preferentially distributed in 3' untranslated regions of cellular transcripts, while the lost m6 A modifications induced by EBV infection preferentially distributed in coding sequence regions of mRNAs. EBV infection could influence the host cellular m6 A epitranscriptome. CONCLUSIONS: These results reveal the critical role of m6 A modification in the process of de novo EBV infection.

BHRF1, a BCL2 viral homolog, disturbs mitochondrial dynamics and stimulates mitophagy to dampen type I IFN induction.

Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-ß induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle's balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Epstein-Barr virus microRNAs in the pathogenesis of human cancers.

The Epstein-Barr Virus (EBV) is a gamma-herpesvirus involved with a variety of human cancers, notably the endemic Burkitt lymphoma and nasopharyngeal carcinoma. In 2004, EBV was described as one the first known human oncoviruses to encode viral microRNAs (miRNAs), and these molecules were found to interact with viral and host targets. EBV miRNAs modulate biological processes that are critical for carcinogenesis, contributing to cell transformation and tumor progression of EBV-associated cancers. Herein we review and discuss EBV miRNAs as modulators of viral biology and carcinogenesis, as well as their usefulness as putative markers to monitor the onset, progression, and recurrence of cancers associated with the EBV infection.

BHRF1 Enhances EBV Mediated Nasopharyngeal Carcinoma Tumorigenesis through Modulating Mitophagy Associated with Mitochondrial Membrane Permeabilization Transition.

Epstein-Barr virus (EBV) is a major contributor to nasopharyngeal carcinoma (NPC) tumorigenesis. Mitochondria have been shown to be a target for tumor viral invasion, and to mediate viral tumorigenesis. In this study, we detected that mitochondrial morphological changes in tumor tissues of NPC patients infected with EBV were accompanied by an elevated expression of BHRF1, an EBV encoded protein homologue to Bcl-2. High expression of BHRF1 in human NPC cell lines enhanced tumorigenesis and metastasis features. With BHRF1 localized to mitochondria, its expression induced cyclophlin D dependent mitochondrial membrane permeabilization transition (MMPT). The MMPT further modulated mitochondrial function, increased ROS production and activated mitophagy, leading to enhanced tumorigenesis. Altogether, our results indicated that EBV-encoded BHRF1 plays an important role in NPC tumorigenesis through regulating cyclophlin D dependent MMPT.

EBV-miR-BHRF1-1 Targets p53 Gene: Potential Role in Epstein-Barr Virus Associated Chronic Lymphocytic Leukemia.

PURPOSE: The purpose of this study was to investigate the prognostic impact of Epstein-Barr virus (EBV)-microRNA (miRNA, miR)-BHRF1-1 with chronic lymphocytic leukemia (CLL) as well as role of EBV-miR-BHRF1-1 in p53 gene. MATERIALS AND METHODS: Quantitative reverse transcription-polymerase chain reaction and western blotting were used to quantify EBV-miR-BHRF1-1 and p53 expression in cultured CLL. RESULTS: p53 aberration was associated with the higher expression level of EBV-miR-BHRF1-1 (p < 0.001) which was also an independent prognostic marker for overall survival (p=0.028; hazard ratio, 5.335; 95% confidence interval, 1.193 to 23.846) in 97 newly-diagnosed CLL patients after adjusted with International Prognostic Index for patients with CLL. We identified EBV-miR-BHRF1-1 as a viral miRNA regulator of p53. EBV-miR-BHRF1-1 repressed luciferase reporter activity by specific interaction with the seed region within the p53 3'- untranslated region. Discordance of p53 messenger RNA and protein expression was associated with high EBV-miR-BHRF1-1 levels in CLL patients and cell lines. EBV-miR-BHRF1- 1 inhibition upregulated p53 protein expression, induced cell cycle arrest and apoptosis and decreased cell proliferation in cell lines. EBV-miR-BHRF1-1 mimics downregulated p53 protein expression, decreased cell cycle arrest and apoptosis, and induced cell proliferation in cell lines. CONCLUSION: This study supported the role of EBV-miR-BHRF1-1 in p53 regulation in vitro. Our results support the potential of EBV-miR-BHRF1-1 as a therapeutic target in EBV-associated CLL with p53 gene aberration.

Pathobiologic Roles of Epstein-Barr Virus-Encoded MicroRNAs in Human Lymphomas.

Epstein-Barr virus (EBV) is a human γ-herpesvirus implicated in several human malignancies, including a wide range of lymphomas. Several molecules encoded by EBV in its latent state are believed to be related to EBV-induced lymphomagenesis, among which microRNAs-small RNAs with a posttranscriptional regulating role-are of great importance. The genome of EBV encodes 44 mature microRNAs belonging to two different classes, including BamHI-A rightward transcript (BART) and Bam HI fragment H rightward open reading frame 1 (BHRF1), with different expression levels in different EBV latency types. These microRNAs might contribute to the pathogenetic effects exerted by EBV through targeting self mRNAs and host mRNAs and interfering with several important cellular mechanisms such as immunosurveillance, cell proliferation, and apoptosis. In addition, EBV microRNAs can regulate the surrounding microenvironment of the infected cells through exosomal transportation. Moreover, these small molecules could be potentially used as molecular markers. In this review, we try to present an updated and extensive view of the role of EBV-encoded miRNAs in human lymphomas.

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis.

The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (∆123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3'UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of pri-miR-BHRF1-2 and 1-3 in the ∆123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the ∆123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis.

Analysis of Viral and Cellular MicroRNAs in EBV-Infected Cells.

MicroRNAs are small, noncoding RNAs that posttranscriptionally regulate gene expression. The discovery of this relatively new mode of gene regulation as well as studies showing the prognostic value of viral and cellular miRNAs as biomarkers, such as in cancer progression, has stimulated the development of many methods to characterize miRNAs. EBV encodes 25 viral precursor microRNAs within its genome that are expressed during lytic and latent infection. In addition to viral miRNAs, EBV infection induces the expression of specific cellular oncogenic miRNAs, such as miR-155, miR-146a, miR-21, and others, that can contribute to the persistence of latently infected cells. This chapter describes several current techniques used to identify and detect the expression of viral and cellular miRNAs in EBV-infected cells.

Epstein-Barr Virus MicroRNAs are Expressed in Patients with Chronic Lymphocytic Leukemia and Correlate with Overall Survival.

Although numerous studies highlighted the role of Epstein-Barr Virus (EBV) in B-cell transformation, the involvement of EBV proteins or genome in the development of the most frequent adult leukemia, chronic lymphocytic leukemia (CLL), has not yet been defined. We hypothesized that EBV microRNAs contribute to progression of CLL and demonstrated the presence of EBV miRNAs in B-cells, in paraffin-embedded bone marrow biopsies and in the plasma of patients with CLL by using three different methods (small RNA-sequencing, quantitative reverse transcription PCR [q-RT-PCR] and miRNAs in situ hybridization [miRNA-ISH]). We found that EBV miRNA BHRF1-1 expression levels were significantly higher in the plasma of patients with CLL compared with healthy individuals (p < 0 · 0001). Notably, BHRF1-1 as well as BART4 expression were detected in the plasma of either seronegative or seropositive (anti-EBNA-1 IgG and EBV DNA tested) patients; similarly, miRNA-ISH stained positive in bone marrow specimens while LMP1 and EBER immunohistochemistry failed to detect viral proteins and RNA. We also found that BHRF1-1 plasma expression levels were positively associated with elevated beta-2-microglobulin levels and advanced Rai stages and observed a correlation between higher BHRF1-1 expression levels and shorter survival in two independent patients' cohorts. Furthermore, in the majority of CLL cases where BHRF1-1 was exogenously induced in primary malignant B cells the levels of TP53 were reduced. Our findings suggest that EBV may have a role in the process of disease progression in CLL and that miRNA RT-PCR and miRNAs ISH could represent additional methods to detect EBV miRNAs in patients with CLL.

BHRF1 exerts an antiapoptotic effect and cell cycle arrest via Bcl-2 in murine hybridomas.

Apoptosis has been widely studied in order to find methods to increase the life-span and production performance in large-scale animal cell cultures. The use of anti-apoptotic genes has emerged as an efficient method to reduce apoptosis in a variety of biotechnological relevant cell lines, including CHO and hybridomas, alternatively to small molecule inhibitors. It is already known that expression of BHRF1, an Epstein-Barr virus-encoded early protein homologous to the anti-apoptotic protein Bcl-2, protects hybridoma cells from apoptosis in batch and continuous operation modes resulting in a delay in the cell death process under glutamine starvation conditions. In the present study, the mechanism of action of BHRF1 was investigated in a murine hybridoma cell line. BHRF1 protein was found in the mitochondrial cell fraction both under normal growing conditions and apoptosis-inducing conditions. Remarkably, the expression of the anti-apoptotic gene bcl2 in BHRF1-expressing cells was up-regulated 25-fold compared to mock-transfected controls under apoptosis triggering conditions and its expression correlated with survival of transgenic cultures and cell cycle arrest in G1. Bcl-2 activity was revealed to be crucial for the BHRF1-mediated effect since the addition of specific inhibitors of Bcl-2 (namely HA14-1 and YC-137) resulted in a loss of function of BHRF1-expressing cells under glutamine starvation conditions. Moreover, the interaction of BHRF1 with the pro-apoptotic BH3-only Bim conferred mitochondrial stability to BHRF1 expressing cells under apoptosis-triggering conditions.

Locating Herpesvirus Bcl-2 Homologs in the Specificity Landscape of Anti-Apoptotic Bcl-2 Proteins.

Viral homologs of the anti-apoptotic Bcl-2 proteins are highly diverged from their mammalian counterparts, yet they perform overlapping functions by binding and inhibiting BH3 (Bcl-2 homology 3)-motif-containing proteins. We investigated the BH3 binding properties of the herpesvirus Bcl-2 homologs KSBcl-2, BHRF1, and M11, as they relate to those of the human Bcl-2 homologs Mcl-1, Bfl-1, Bcl-w, Bcl-xL, and Bcl-2. Analysis of the sequence and structure of the BH3 binding grooves showed that, despite low sequence identity, M11 has structural similarities to Bcl-xL, Bcl-2, and Bcl-w. BHRF1 and KSBcl-2 are more structurally similar to Mcl-1 than to the other human proteins. Binding to human BH3-like peptides showed that KSBcl-2 has similar specificity to Mcl-1, and BHRF1 has a restricted binding profile; M11 binding preferences are distinct from those of Bcl-xL, Bcl-2, and Bcl-w. Because KSBcl-2 and BHRF1 are from human herpesviruses associated with malignancies, we screened computationally designed BH3 peptide libraries using bacterial surface display to identify selective binders of KSBcl-2 or BHRF1. The resulting peptides bound to KSBcl-2 and BHRF1 in preference to Bfl-1, Bcl-w, Bcl-xL, and Bcl-2 but showed only modest specificity over Mcl-1. Rational mutagenesis increased specificity against Mcl-1, resulting in a peptide with a dissociation constant of 2.9nM for binding to KSBcl-2 and >1000-fold specificity over other Bcl-2 proteins, as well as a peptide with >70-fold specificity for BHRF1. In addition to providing new insights into viral Bcl-2 binding specificity, this study will inform future work analyzing the interaction properties of homologous binding domains and designing specific protein interaction partners.

Epstein-Barr virus (EBV) association and latency profile in pediatric Burkitt's lymphoma: experience of a single institution in Argentina.

The aim of this study is to characterize EBV expression and latency pattern in pediatric Burkitt's lymphoma in a single institution in Argentina. EBV-encoded RNA or protein was analyzed in 27 patients. EBERs was expressed in 37% of patients (29% of immunocompetent and 100% of immunosuppressed patients). EBV-positive cases were observed exclusively in patients younger than 5 years old. EBV association with immunocompetent patients exhibits the sporadic pattern in region under study, while its presence in patients infected with HIV was higher than described previously. EBV latency I profile was present in most of the patients, except for two immunosuppressed patients who displayed LMP1 expression.

Macroautophagy Proteins Assist Epstein Barr Virus Production and Get Incorporated Into the Virus Particles.

Epstein Barr virus (EBV) persists as a latent herpes virus infection in the majority of the adult human population. The virus can reactivate from this latent infection into lytic replication for virus particle production. Here, we report that autophagic membranes, which engulf cytoplasmic constituents during macroautophagy and transport them to lysosomal degradation, are stabilized by lytic EBV replication in infected epithelial and B cells. Inhibition of autophagic membrane formation compromises infectious particle production and leads to the accumulation of viral DNA in the cytosol. Vice versa, pharmacological stimulation of autophagic membrane formation enhances infectious virus production. Atg8/LC3, an essential macroautophagy protein and substrate anchor on autophagic membranes, was found in virus preparations, suggesting that EBV recruits Atg8/LC3 coupled membranes to its envelope in the cytosol. Our data indicate that EBV subverts macroautophagy and uses autophagic membranes for efficient envelope acquisition during lytic infection.