Search

Abstract Background Immunotherapy has revolutionized the therapeutical regimen for nasopharyngeal carcinoma (NPC), yet its response rate remains insufficient. Programmed death‐ligand 1 (PD‐L1) on small extracellular vesicles (sEVs) mediates local and peripheral immunosuppression in tumors, and the mechanism of PD‐L1 loading into these vesicles is garnering increasing attention. Latent membrane protein 1 (LMP1), a key viral oncoprotein expressed in Epstein‐Barr virus (EBV)‐positive NPC, contributes to remodeling the tumor microenvironment. However, the precise mechanisms by which LMP1 modulates tumor immunity in NPC remain unclear. Here, we aimed to investigate the roles and regulatory mechanisms of LMP1 and sEV PD‐L1 in NPC immune evasion. Methods We analyzed the impact of LMP1 on tumor‐infiltrating lymphocyte abundance in NPC tissues and humanized tumor‐bearing mouse models using multiplex immunofluorescence (mIF) and flow cytometry, respectively. Transmission electron microscopy and nanoparticle tracking analysis were employed to characterize sEVs. Immunoprecipitation‐mass spectrometry was utilized to identify proteins interacting with LMP1. The regulatory effects of sEVs on tumor microenvironment were assessed by monitoring CD8+ T cell proliferation and interferon‐γ (IFN‐γ) expression via flow cytometry. Furthermore, the expression patterns of LMP1 and downstream regulators in NPC were analyzed using mIF and survival analysis. Results High LMP1 expression in NPC patient specimens and mouse models was associated with restricted infiltration of CD8+ T cells. Additionally, LMP1 promoted sEV PD‐L1 secretion, leading to inhibition of CD8+ T cell viability and IFN‐γ expression in vitro. Mechanistically, LMP1 recruited apoptosis‐linked gene 2‐interacting protein X (ALIX) through its intracellular domain and bound PD‐L1 through its transmembrane domain, thereby facilitating the loading of PD‐L1 into ALIX‐dependent sEVs. Disruption of ALIX diminished LMP1‐induced sEV PD‐L1 secretion and enhanced the anti‐tumor immunity of CD8+ T cells both in vitro and in vivo. Moreover, increased expression levels of LMP1 and ALIX were positively correlated with enhanced immunosuppressive features and worse prognostic outcomes in NPC patients. Conclusion Our findings uncovered the mechanism by which LMP1 interacts with ALIX and PD‐L1 to form a trimolecular complex, facilitating PD‐L1 loading into ALIX‐dependent sEV secretion pathway, ultimately inhibiting the anti‐tumor immune response in NPC. This highlights a novel target and prognostic marker for NPC immunotherapy.

Epstein-Barr virus (EBV) infection has a strong correlation with the development of nasopharyngeal carcinoma (NPC). Aquaporin 3 (AQP3), a member of the aquaporin family, plays an important role in tumor development, especially in epithelial-mesenchymal transition. In this study, the expression of AQP3 in EBV-positive NPC cells was significantly lower than that in EBV-negative NPC cells. Western blot and qRT-PCR analysis showed that LMP1 down-regulated the expression of AQP3 by activating the ERK pathway. Cell biology experiments have confirmed that AQP3 affects the development of tumor by promoting cell migration and proliferation in NPC cells. In addition, AQP3 can promote the lysis of EBV in EBV-positive NPC cells. The inhibition of AQP3 expression by EBV through LMP1 may be one of the mechanisms by which EBV maintains latent infection-induced tumor progression. [ABSTRACT FROM AUTHOR]

Introduction: Endometriosis (EMS) is characterized as a prevalent gynecological inflammatory disorder marked by the existence of endometrial tissues situated beyond the uterus. This condition leads to persistent pelvic pain and may contribute to infertility. In this investigation, we explored the potential mechanism underlying the development of endometriosis (EMS) triggered by transient exposure to either latent membrane protein 1 (LMP1) or Epstein-Barr virus (EBV) in a mouse model. Additionally, we examined the potential inhibitory effect of evodiamine (EDM) on EMS. Methods: Immortalized human endometrial stromal cells (HESC) or epithelial cells (HEEC) were transiently exposed to either EBV or LMP1. The presence of evodiamine (EDM) was assessed for its impact on estrogen receptor β (ERβ) expression, as well as on cell metabolism parameters such as redox balance, mitochondrial function, inflammation, and proliferation. Additionally, a mixture of LMP1-treated HESC and HEEC was administered intraperitoneally to generate an EMS mouse model. Different dosages of EDM were employed for treatment to evaluate its potential suppressive effect on EMS development. Results: Transient exposure to either EBV or LMP1 triggers persistent ERβ expression through epigenetic modifications, subsequently modulating related cell metabolism for EMS development. Furthermore, 4.0 μM of EDM can efficiently reverse this effect in in vitro cell culture studies. Additionally, 20 mg/kg body weight of EDM treatment can partly suppress EMS development in the in vivo EMS mouse model. Conclusion: Transient EBV/LMP1 exposure triggers permanent ERβ expression, favoring later EMS development, EDM inhibits EMS development through ERβ suppression. This presents a novel mechanism for the development of endometriosis (EMS) in adulthood stemming from early Epstein-Barr virus (EBV) exposure during childhood. Moreover, evodiamine (EDM) stands out as a prospective candidate for treating EMS. [ABSTRACT FROM AUTHOR]

Hodgkin lymphoma is histologically characterised by the presence of Hodgkin (H) and Reed‐Sternberg (RS) cells originating from germinal centre B‐cells rearranged in the IgV gene. The formation of multinucleated RS cells is a product of telomere organisation in a process initiated by telomere aggregate accumulation in mononuclear H cells and may be mediated by latent membrane protein 1 (LMP‐1) expression. LMP‐1 is the main oncoprotein of EBV and supports several tumourigenic processes. LMP‐1 may rescue proapoptotic B‐cells through downregulation of B‐cell receptor (BCR) components, mimicking and inducing multiple distinct B‐cell signalling pathways to promote proliferation and survival, such as Janus kinase‐signal transducer and activator of transcription (JAK‐STAT), nuclear factor‐kappa b (NF‐кB), and cellular MYC (c‐MYC), and inducing telomere instability mainly through Telomere repeat binding factor 2 (TRF2) downregulation to promote the formation of multinucleated RS cells. This review presents recent discoveries regarding the influence of LMP‐1 on the surviving cellular signalling, genomic instability and mecanical formation of HRS cells. [ABSTRACT FROM AUTHOR]

The disruption of antiviral sensors and the evasion of immune defences by various tactics are hallmarks of EBV infection. One of the EBV latent gene products, LMP1, was shown to induce the activation of signalling pathways, such as NF-κB, MAPK (JNK, ERK1/2, p38), JAK/STAT and PI3K/Akt, via three subdomains of its C-terminal domain, regulating the expression of several cytokines responsible for modulation of the immune response and therefore promoting viral persistence. The aim of this review is to summarise the current knowledge on the EBV-mediated induction of immunomodulatory molecules by the activation of signal transduction pathways with a particular focus on LMP1-mediated mechanisms. A more detailed understanding of the cytokine biology molecular landscape in EBV infections could contribute to the more complete understanding of diseases associated with this virus. [ABSTRACT FROM AUTHOR]

Rationale. The discovery of two Epstein–Barr virus (EBV) types, EBV-1 and EBV-2, which differ in their transforming abilities stimulated their study in various populations in order to elucidate the relationship with malignant neoplasms.Aim. To study EBV-1 and EBV-2 in representatives of four ethnic groups of Russia, sequence analysis of the LMP1 oncogene in virus isolates, and search for a correlation between the types of EBV and the incidence of certain types of malignant tumors in four ethnic groups.Materials and methods. From total DNA obtained from oral washings of Adygeans, Kalmyks, Tatars and Slavs, representatives of 3 Russian republics and the Moscow region, EBV DNA was amplified; its presence and concentration were analyzed by nested PCR, and each product was examined for the virus type and LMP1 variant. Types of EBV found in representatives of In four ethnic groups were compared with the incidence rates of malignant tumors, among which EBV-associated cases may occur, in population of three republics and the Moscow region.Results. It was observed that higher gastric cancer and lymphomas morbidity among Tatarstan and Moscow region populations (compared to populations of Adygea and Kalmykia) correlated with predominance in these ethnic groups – Tatars and Slavs – EBV-1 type having transforming potential in vitro. In Adygeis, non-transforming EBV-2 type was prevalent, and Kalmyks were infected with both types in approximately equal proportion. however, differences between tumor morbidities between the compared groups were statistically insignificant (p >0.05). The identified spectrum of LMP1 variants, as well as the results of their sequencing, show genetic relations between EBV strains circulating in the studied ethnic groups.Conclusion. Study of EBV isolates for the first time performed in 4 ethnic groups of this country showed differences in distribution of the types of this virus but there was no correlation with tumor morbidity.

Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic or recurrent infectious mononucleosis-like symptoms and is associated with EBV-associated T/natural killer (NK)-cell lymphoproliferative disorders, which frequently lead to the development of life-threatening complications, such as virus-associated hemophagocytic syndrome and EBV-positive apparent leukemia/lymphoma mainly in T- and NK-cell lineages. In order to clarify the EBV genes responsible for the diseases, we introduced the plasmid coding sequences of EBV-encoded small RNAs (EBERs) and/or latent membrane protein (LMP) 1 into human T-lymphocyte virus-I-negative human T-cell lines using a gene expression vector harboring EBV nuclear antigen 1, established the G418-resistant transformants of five T-cell lines, and quantitatively examined the expression of EBERs and LMP1 using real-time reverse transcriptase–polymerase chain reaction. The expression levels of EBERs in T-cell transformants with EBER DNA paralleled those in EBV-positive human T- and NK-cell lines, SNTK cells. The expression of LMP1 mRNA varied in SNTK cells and in human T-cell transformants, and the expression of LMP1 mRNA in T-cell lines expressing both EBERs and LMP1 was much lower than that in the same cell line expressing LMP1 mRNA alone. The currently employed gene expression system and currently obtained transformants may be useful for the analyses of the pathophysiology of CAEBV and EBV-positive T/NK-cell lymphoproliferative disorders. [ABSTRACT FROM AUTHOR]

Background: Stromal interaction molecule 1 (STIM1)-mediated Ca2+ signaling regulates tumor angiogenesis in nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-related human malignancy. However, the mechanism by which STIM1 modulates endothelial functional phenotypes contributing to tumor angiogenesis remains elusive. Methods: NPC cell-derived exosomes were isolated via differential centrifugation and observed using transmission electron microscopy. Exosome particle sizes were assessed by nanoparticle tracking analysis (NTA). Uptake of exosomes by recipient ECs was detected by fluorescent labeling of the exosomes with PKH26. Tumor angiogenesis-associated profiles were characterized by determining cell proliferation, migration, tubulogenesis and permeability in human umbilical vein endothelial cells (HUVECs). Activation of the Akt/ERK pathway was assessed by detecting the phosphorylation levels using Western blotting. A chick embryo chorioallantoic membrane (CAM) xenograft model was employed to study tumor-associated neovascularization in vivo. Results: We found that NPC cell-derived exosomes harboring EBV-encoded latent membrane protein 1 (LMP1) promoted proliferation, migration, tubulogenesis and permeability by activating the Akt/ERK pathway in ECs. STIM1 silencing reduced LMP1 enrichment in NPC cell-derived exosomes, thereby reversing its pro-oncogenic effects in an Akt/ERK pathway-dependent manner. Furthermore, STIM1 knockdown in NPC cells blunted tumor-induced vascular network formation and inhibited intra-tumor neovascularization in the chorioallantoic membrane (CAM) xenograft model. Conclusion: STIM1 regulates tumor angiogenesis by controlling exosomal EBV-LMP1 delivery to ECs in the NPC tumor microenvironment. Blocking exosome-mediated cell-to-cell horizontal transfer of EBV-associated oncogenic signaling molecules may be an effective therapeutic strategy for NPC. [ABSTRACT FROM AUTHOR]

Aim: To investigate the role of AHR in nasopharyngeal carcinoma (NPC) and explore the relationship between Epstein–Barr virus (EBV) infection and the AHR pathway. Methods: The effect of LMP1 on the expression of AHR was analyzed using real-time PCR and western blot. Proliferation and migration of cells were assessed using CCK8 and Transwell analysis. Results: EBV infection downregulated the expression of AHR in NPC cells, possibly through activation of the ERK pathway by LMP1 thereby accelerating AHR proteasomal degradation following translocation to the nucleus. Cell proliferation, migration and autophagy are promoted by activating the AHR pathway. Conclusion: In NPC cells, LMP1 increases the phosphorylation of ERK, which may activate the AHR pathway. This article describes the role of a virus, the Epstein–Barr virus (EBV), in the development of a type of cancer that affects the throat called nasopharyngeal carcinoma (NPC). One of the proteins made by the virus, called latent membrane protein 1 (LMP1), is thought to play a role in the development of NPC. We investigate how LMP1 may cause NPC. We found that LMP1 was linked to higher levels of two genes, CYP1A1 and CYP1B1, which are targets of a receptor called the aryl hydrocarbon receptor (AHR). This may be linked to the movement of AHR to the cell's control center, where it can play a role in the development of cancer. This article suggests a pathway for how EBV may cause NPC. [ABSTRACT FROM AUTHOR]

Introduction. It is known that the structural features of the Epstein-Barr virus (EBV) affect the manifestation of its biological properties. Based on differences in the sequences of the EBNA2, EBNA3A, -B, and -C genes, two types of the virus, EBV-1 and EBV-2, have been identified that have different ability to transform B cells in vitro and possibly playing certain role in the development of EBV-associated neoplasms.Aim. To study the prevalence of EBV-1 and EBV-2 in two ethnic groups, Аdygeans and Slavs, as well as the contribution of EBV-associated tumors to the overall incidence of malignant neoplasms certain organs and tissues.Materials and methods. DNA samples were extracted from 59 oral lavages of ethnic Аdygeans from Republic of Adygea and 40 such from oral cavity of ethnic Slavs of Moscow city. These samples were used for amplification of EBV DNA, determination of the concentration of viral DNA copies per 1 cell washout, as well as for amplification of EBV LMP1 followed by sequencing of the resulting gene samples and determination of their protein variant (LMP1).Results. Studies have shown that among the representatives of the Аdygeans the 2nd EBV type prevails, and among the Slavs, the 1st one. Epstein-Barr virus isolates in representatives of the two ethnic groups also differed in the structure of LMP1. Among the Slavs, a set of its LMP1 variants (B95.8/A, China, Med- and NC) was identified. However, among the Adygeans, the only variant - B95.8 and its subtype - B95.8/A was identified. EBV-1, which prevails among the representatives of the Slavs and has the ability to transform B-cells, was projected onto a higher incidence of tumors of the pharynx, stomach, Hodgkin's and non-Hodgkin's lymphomas (where EBV-associated cases cam occur) in the population of Moscow than in the population of the Republic of Adygea. However, the differences between incidence rates for these neoplasms (with the exception for the stomach tumors) were not statistically significant (p >0.5). A higher and statistically significantly different incidence rate of stomach cancer in residents of Moscow city, compared with that in residents of the Republic of Adygea, in our opinion, is not due to EBV-1 type and/or LMP1 variants, but rather is associated with a genetic predisposition the population of Moscow city to this tumor.Conclusion. The fact that two ethnic groups of Russia were found to be prevails by different types of EBV raises the question of their ethno-geographical association and their role in the induction of EBV-associated tumors. To resolve this issue additional studies in other geographical regions of Russia among representatives of different ethnic groups are required.

Corroles have attracted increasing research interests in recent decades owing to their unique properties over porphyrins. However, the relatively inefficient and tedious synthetic procedures of corrole building blocks with functional groups for bioconjugation hindered their bioapplications. Herein, we report a highly efficient protocol to synthesize corrole‐peptide conjugates with good yields (up to 63 %) without using prepared corrole building blocks. By condensing two −COOH‐bearing‐dipyrromethane molecules onto an aldehyde group on resin‐bound peptide chains in a controllable manner, a series of desired products with long (up to 25 residues) and bioactive peptide chains were obtained with at most one chromatographic purification. The synthesized compounds exhibited potential applications as chelators for metal ions for biomedical applications, as building blocks for supramolecular materials, as well as targeted fluorescent probes. [ABSTRACT FROM AUTHOR]

Invadopodia are actin‐rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they have been visualized as localized and dot‐like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein–Barr virus (EBV)‐associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live‐cell imaging and molecular assays revealed the interaction of macrophage‐released TNFα and EBV‐encoded latent membrane protein 1 (LMP1) in co‐activating the EGFR/Src/ERK/cortactin and Cdc42/N‐WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot‐like digestion patterns on a 2D gelatin to a dendrite‐like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot‐like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα‐rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic or recurrent infectious mononucleosis-like symptoms and is associated with EBV-associated T/natural killer (NK)-cell lymphoproliferative disorders, which frequently lead to the development of life-threatening complications, such as virus-associated hemophagocytic syndrome and EBV-positive apparent leukemia/lymphoma mainly in T- and NK-cell lineages. In order to clarify the EBV genes responsible for the diseases, we introduced the plasmid coding sequences of EBV-encoded small RNAs (EBERs) and/or latent membrane protein (LMP) 1 into human T-lymphocyte virus-I-negative human T-cell lines using a gene expression vector harboring EBV nuclear antigen 1, established the G418-resistant transformants of five T-cell lines, and quantitatively examined the expression of EBERs and LMP1 using real-time reverse transcriptase–polymerase chain reaction. The expression levels of EBERs in T-cell transformants with EBER DNA paralleled those in EBV-positive human T- and NK-cell lines, SNTK cells. The expression of LMP1 mRNA varied in SNTK cells and in human T-cell transformants, and the expression of LMP1 mRNA in T-cell lines expressing both EBERs and LMP1 was much lower than that in the same cell line expressing LMP1 mRNA alone. The currently employed gene expression system and currently obtained transformants may be useful for the analyses of the pathophysiology of CAEBV and EBV-positive T/NK-cell lymphoproliferative disorders.

Background: Cancers of the upper aero-digestive tract (UADT) are fifth most common cancer in the world with around 10,55,000 new cases and 7,25,000 deaths worldwide. Tobacco and alcohol act synergistically and are the two most important etiological factors responsible for about 75% of SCC. Studies have reported changing trends in the incidence of SCC showing an increasing shift in epidemiology attributed to the infection by viruses. The most commonly implicated viruses are the Human Papillomavirus (HPV) and Epstein-Barr virus (EBV). Aim and Objective: To study association of Human Papilloma virus (HPV) and Epstein Barr virus (EBV) with 100 newly diagnosed cases of squamous cell carcinoma (SCC) of upper aerodigestive tract (UADT) and correlate its association with clinical parameters, histomorphological grade and staging using immunohistochemical markers p16, LMP1, p53, p63. Materials and Methods: The study was conducted in the department of Pathology, Maulana Azad medical college and associated hospitals, New Delhi from September 2018-April 2019. Specimens was routinely processed. Immunohistochemistry (IHC) was done using p16, LMP1, p53, p63 monoclonal antibodies. Statistical Analysis Used: SPSS 20 software. The quantitative analysis was done using Pearson chi-square test. Probability values < 0.05 was be considered statistically significant. Results: HPV was present in 29% cases while EBV in 38% cases. Oral cavity was the most common site involved by both HPV and EBV. Co-infectivity was found in 4% cases. There was significant male predominance in both. HPV was more prevalent in age >45 years while EBV was equally distributed in <45 years and >45 years. Moderately differentiated squamous cell carcinoma was the commonest grade involved by both the viruses. A significant correlation was found between EBV and alcohol. p53 positivity had an inverse relationship with HPV positivity. P63 expression was higher in HPV and EBV positive cases. Conclusion: In resource constraint settings, p16 and Latent membrane protein 1 can be used as surrogacy markers for Human Papilloma virus and Epstein Barr virus along with p53 and p63 for its association with histomorphological grade and stage. [ABSTRACT FROM AUTHOR]

Aim: The current study aimed to investigate sequence variations in the C-terminus of latent membrane protein 1 (LMP1) in Epstein- Barr virus (EBV) isolates from Iranian patients with chronic gastritis or gastric cancer (GC). Background: LMP1, an essential viral oncoprotein, is the critical element in the immortalization of B cells. It contains a small twenty-four amino acid cytoplasmic N-terminal region, six transmembrane segments, and a two hundred amino acid cytoplasmic Cterminal domain. Most LMP1-mediated signal transduction events are moderated by some functional parts of the cytoplasmic Cterminal domain. Methods: Thirty-two EBV-positive biopsy tissues were obtained from patients with gastric cancer and patients with chronic gastritis. The C-terminal nucleotide sequences of LMP1 were amplified using nested-PCR and analyzed by DNA sequencing. Results Four to eight copies of the 11 repeat elements (codon 254--302) were observed in the carboxyl-terminal site of patients, but no relationship was found between the number of repeat sequences and disease status. The 30-bp deletion corresponding to codon 345--354 of the B95-8 strain was observed in 34% of isolates, and the remaining samples were non-deleted. In the gastric cancer group, a higher number of 33-bp repeats (≥5 repeats) was observed in 30-bp-deletion (100%) than in non-deleted (42%) isolates, and the difference was statistically significant. Analysis revealed that a gastritis isolate may be the result of recombination between Alaskan and China1 strains. Conclusion: Overall, the current results showed no association between C-terminal sequence variations of LMP1 and malignant or non-malignant isolate origin. [ABSTRACT FROM AUTHOR]

Objective: To compare genetic structure of the main Epstein–Barr virus (EBV) oncogene, latent membrane protein 1 (LMP1), in EBV strains circulating in two genetically distinct ethnic populations in Russian Federation, Tatars and Slavs, as well as assess an impact of diverse LMP1 variants on incidence and mortality rate for some malignant tumors partially associated with EBV infection. Materials and methods. Oral washing samples were collected from 60 ethnic Kazan Tatars and 65 ethnic Moscow Slavics. Carboxy-terminal nucleotide sequences (41 and 40 sequences, respectively) derived from hypervariable LMP1 gene region were amplified from EBV DNA samples. Next, final nucleotide sequences were translated into amino acid sequences and analyzed according to classification by Edwards et al. Results. Analysis of 41 and 40 LMP1 samples obtained from ethnic Kasan Tatars and ethnic Moscow Slavics, respectively, revealed significant difference in relevant amino acid structures. In particular, all LMP1 samples derived from Moscow Slavics were found to belong to the four protein variants: B95.8/A, Med–, China1 and NC. Among them, low-transforming variant B95.8/A was dominant (82.5%). In contrast, solely 21 out of 41 LMP1 samples derived from ethnic Tatars were classified as B95.8/A, Med– and China1 variants. Importantly, the percentage of low-transforming B95.8/A variant among ethnic Tatar samples was significantly lower compared to that one found in Moscow Slavics (29.3% vs. 82.5%). On the other hand, seven (17.1%) out of 20 other samples formed a unique protein mono group characterized by LMP1 amino acid sequence differed from that one available in the GenBank database. Such group of variants was designated as LMP1-TatK. The remaining 13 samples (31.7%) did not match either protein variants, thereby forming the “beyond classification” (LMP1-TatBC) group. Conclusion. The data obtained suggest that various LMP1 variants exist in EBV strains persisting in ethnic Tatrs and ethnic Slavics examined in Russian Federation. It was also found that EBV strains isolated from ethnic Tatars contained a unique LMP1 gene variant encoding protein LMP1-TatK lacked in EBV strains derived from ethnic Moscow Slavics. Taking into account the genealogy of Tatars, it cannot be ruled out that EBV strain bearing LMP1-TatK variant represented ethnically specific EBV strain that might circulate many centuries ago among their historical human predecessors called Mongol-Tatar tribes. In addition, it was shown that the LMP1 variants in EBV strains isolated from ethnic Kazan Tatars and ethnic Moscow Slavics did not affect the incidence and mortality of different forms of cancer consisting of EBV-associated cases.

Introduction. Molecular studies have shown that viruses appeared in the early stages of the evolution of life. For millions of years, viruses have evolved by changing old and acquiring new sequences in their RNA or DNA. It is assumed that most viruses have common ancestors. Such an ancestor, an ancient strain, probably existed for Epstein-Barr virus (EBV) as well. Aim. To find out whether ancient strains of EBV persist in modern Russian ethnic groups today. Material and methods. The object of the study was the EBV LMP1oncogene, which is most suitable for molecular genetic analysis. LMP1 was amplified from the oral cavity washings obtained from representatives of two ancient ethnic groups of Russia - Tatars and Slavs. The LMP1 amplicons were sequenced in both directions; their nucleotide sequences translated into amino acid (LMP1) were evaluated using the classification suggested by Edwards et al. 1999. To establish genetic relationships between LMP1 variants, a phylogenetic tree was constructed by the neighbor-joining method using the MEGA software package. Results and discussion. Analysis of LMP1 sequences from washings of the Slavs oral cavity demonstrated the presence of LMP1 variants with varying degrees of transforming potential: B98.5/A, China1, Med-, and NC. The analysis of LMP1 sequences from washings of Tatar oral cavity also made it possible to identify oncoprotein variants such as B95.8/A, China1, Med-, as well as a group of variants out of classifications (LMP1-OK). An important finding was the identification of 7 variants of LMP1 from Tatars, designated as LMP1-TatK, that contained two unique deletions of 5 aa in codons 312-316 and 382-386, which were absent in the LMP1 variants from Slavs and from previously examined cancer patients and healthy individuals, as well as in sequences from open computer databases. The uniqueness of the LMP1-TatK variant is confirmed both by phylogenetic analysis of LMP1 sequences of Tatar origin and by the analysis of 11 aa repeats and 5 aa insertions in the C-terminal region of the oncoprotein. The morbidity and mortality rates from neoplasms, including EBV-associated pathologies, did not differ significantly between two studied ethnic groups infected with different EBV strains. Conclusion. The data obtained allowed us to suggest that: 1) LMP1-TatK could be refered to an evolutionarily ancient EBV strain that persists among Tatars and; 2) LMP1-TatK belongs to the so-called “Volga” EBV virus strain, the common strain among the population of the Volga region. Extended studies of EBV isolates from residents of this region may probably shed the light on the origin of LMP1-TatK.

Natural killer/T-cell lymphoma (NKTCL) is an aggressive Epstein-Barr virus (EBV)-associated non-Hodgkin lymphoma with poor prognosis. In this study, we aimed to investigate the potential mechanism of latent membrane protein 1 (LMP1)-mediated tumorigenesis and provide a novel therapeutic strategy for targeting the EBV DNA genome. We found that LMP1 upregulated the expression of peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1β (PGC1β) through activation of nuclear factor-κB (NF-κB). Furthermore, the activated PGC1β upregulated the expression of 8-oxoguanine DNA glycosylase (OGG1) through the coactivation of nuclear respiratory factor 1 (NRF1) and GA-binding protein α (GABPα), preventing reactive oxygen species (ROS)-mediated base incision in the EBV genome and favoring its survival. Interruption of hexokinase domain component 1 (HKDC1) by either shRNA or Tf-D-HKC8 peptide suppressed the interaction of HKDC1 with voltage-dependent anion channel 1 (VDAC1), triggering mitochondrial dysfunction and excessive generation of ROS, thus resulting in EBV suppression through ROS-mediated DNA damage. Suppression of the EBV genome inhibited the expression of the LMP1/PGC1β/HKDC1/OGG1 signaling pathway, forming a positive feed forward loop for the generation of ROS, hence inhibiting the EBV genome and subsequent EBV-associated tumor development. We concluded that LMP1 triggers EBV-associated tumorigenesis through activation of the PGC1β pathway. This study provided a novel therapeutic strategy for the treatment of EBV-associated tumors by targeting HKDC1. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV)—the prototypical human tumor virus—is responsible for 1–2% of the global cancer burden, but divergent strains seem to exist in different geographical regions with distinct predilections for causing lymphoid or epithelial malignancies. Here we report the establishment and characterization of Yu103, an Asia Pacific EBV strain with a highly remarkable provenance of being derived from nasopharyngeal carcinoma biopsy but subsequently propagated in human B-lymphoma cells and xenograft models. Unlike previously characterized EBV strains which are either predominantly B-lymphotropic or epitheliotropic, Yu103 evinces an uncanny capacity to infect and transform both B-lymphocytes and nasopharyngeal epithelial cells. Genomic and phylogenetic analyses indicated that Yu103 EBV lies midway along the spectrum of EBV strains known to drive lymphomagenesis or carcinogenesis, and harbors molecular features which likely account for its unusual properties. To our knowledge, Yu103 EBV is currently the only EBV isolate shown to drive human nasopharyngeal carcinoma and B-lymphoma, and should therefore provide a powerful novel platform for research on EBV-driven hematological and epithelial malignancies.

Epstein-Barr virus (EBV)—the prototypical human tumor virus—is responsible for 1–2% of the global cancer burden, but divergent strains seem to exist in different geographical regions with distinct predilections for causing lymphoid or epithelial malignancies. Here we report the establishment and characterization of Yu103, an Asia Pacific EBV strain with a highly remarkable provenance of being derived from nasopharyngeal carcinoma biopsy but subsequently propagated in human B-lymphoma cells and xenograft models. Unlike previously characterized EBV strains which are either predominantly B-lymphotropic or epitheliotropic, Yu103 evinces an uncanny capacity to infect and transform both B-lymphocytes and nasopharyngeal epithelial cells. Genomic and phylogenetic analyses indicated that Yu103 EBV lies midway along the spectrum of EBV strains known to drive lymphomagenesis or carcinogenesis, and harbors molecular features which likely account for its unusual properties. To our knowledge, Yu103 EBV is currently the only EBV isolate shown to drive human nasopharyngeal carcinoma and B-lymphoma, and should therefore provide a powerful novel platform for research on EBV-driven hematological and epithelial malignancies. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between hematologic tumors and Epstein-Barr virus (EBV)-encoded small noncoding RNA (EBER) variations as well as latent membrane protein 1 (LMP1) variations. Methods: Patients with leukemia and myelodysplastic syndrome (MDS) were selected as subjects. Genotypes 1/2 and genotypes F/f were analyzed using the nested PCR technology, while EBER and LMP1 subtypes were analyzed by the nested PCR and DNA sequencing. Results: Type 1 was more dominant than type 2, found in 59 out of 82 (72%) leukemia and in 31 out of 35 (88.6%) MDS, while type F was more prevalent than type f in leukemia (83/85, 97.6%) and MDS (29/31, 93.5%) samples. The distribution of EBV genotypes 1/2 was not significantly different among leukemia, MDS, and healthy donor groups, neither was that of EBV genotypes F/f. EB-6m prototype was the dominant subtype of EBER in leukemia and MDS (73.2% [30/41] and 83.3% [10/12], respectively). The frequency of EB-6m was lower than that of healthy people (96.7%, 89/92), and the difference was significant (p < 0.05). China 1 subtype was the dominant subtype of LMP1 in leukemia and MDS (70% [28/40] and 90% [9/10], respectively), and there was no significant difference in the distribution of LMP1 subtypes among the 3 groups (p > 0.05). Conclusion: The distribution of EBV 1/2, F/f, EBER, and LMP1 subtypes in leukemia and MDS was similar to that in the background population in Northern China, which means that these subtypes may be rather region-restricted but not associated with leukemia and MDS pathogenesis. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. It is also characterized by heavy infiltration with non-malignant leucocytes. The EBV-encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signaling pathways which collectively promote cell proliferation and survival, angiogenesis, invasiveness, and aerobic glycolysis. LMP1 also affects cell-cell interactions, antigen presentation, and cytokine and chemokine production. Here, we discuss how LMP1 modulates local immune responses that contribute to the establishment of the NPC tumor microenvironment. We also discuss strategies for targeting the LMP1 protein as a novel therapy for EBV-driven malignancies.

The involvement of the central nervous system (CNS) in Hodgkin lymphoma (HL) has been rarely reported, especially in its primary isolated form. Herein, we present a case of a 33-year-old woman, who received immunosuppressive treatment due to ulcerative colitis (at the beginning azathioprine and sulfasalazine, changed to mesalazine), with repetitive episodes of loss of consciousness for a few weeks and with no other symptoms. Magnetic resonance imaging scans of the head revealed a tumor in the lateral part of the left temporal lobe and in the cerebellum. Moreover, a subsequent computed tomographic scan of the chest revealed diffuse tumorous lesions in the lungs. The brain tumor was resected and a tumorous lesion resected from the lungs was biopsied. The histopathological analysis confirmed the final diagnosis of HL localized in the CNS with concomitant pulmonary lymphomatoid granulomatosis (LYG) grade 1. After the patient underwent radiotherapy and chemotherapy, the patient showed complete regression of lesions in the CNS and lungs, which was confirmed by positron emission tomographic scan. LYG and CNS-HL are rare proliferative disease derived from lymphocytes B and associated with EBV infections. An association between LYG and other autoimmune disorders has been reported, but to the best of our knowledge, this is the first case of the CNS-HL associated with lymphatoid granulomatosis.

Objective of the investigation was to study the infection of ethnic Tatars with the Epstein–Barr virus (EBV) and to analyze the genetic structure of the oncogene of the virus, the latent membrane protein 1 (LMP1), in the virus strains of Tatar origin. Materials and methods. The materials for the study were samples of boucle flushes of 60 students from the Kazan State Medical University who are ethnic Tatars (Tatars no less than in the 3rd generation). Amplified from DNA of boucle flushes the nucleotide sequences of the LMP1 samples translated into DNA amino acid sequences, have undergone classification based on the well-known and widely used in literature the R.H. Edwards et al. classification. Results. The analysis of nucleotide and deductive amino acid sequences of the 41 LMP1 amplicons revealed their homology with only three gene variants from the R.H. Edwards et al. classification (1999): 95.8/A (29.3 %; 12/41), Med– (14.6 %; 6/41) and China1 (7.3 %, 3/41). Such variants of LMP1 as Alaskan, Med+, Chinа2, China3 and NC, were not found. Among the LMP1 samples of Tatar origin in 20 cases (48.8 %), the composition of the mutations found did not allow them to be assigned to any of the oncogene variants listed above. Out of this number, in 7 (17.1 %) cases a mono group of LMP1 samples was found, differing not only from representatives of the Slavs, inhabitants of the European part of Russia, but also from other Kazan samples, and was designated as LMP1-TatK. The remaining 13 samples of LMP1 (31.7 %), not belonging to any of the known classifications, formed the group designated by us as an LMP1 group beside the classification (LMP1BC). Conclusion. Continuation of the study of the molecular-biological and functional properties of LMP1 in TatK and BC groups, which constitute 48.8 % of the number of gene samples studied, and an analysis of the peculiarities of the ethnic Tatar genotype, will probably help to clarify whether certain EBV strains influence morbidity and mortality in Tatar population with malignant neoplasms, which include EBVassociated cases.

Abstract Background Natural killer/T-cell lymphoma (NKTCL) is a highly aggressive non-Hodgkin lymphoma often resistant to chemotherapy. Serum level of soluble IL-2 receptor α (IL-2Rα) is elevated in NKTCL patients and correlates significantly with treatment response and survival. In the current study we examined the potential role of IL-2Rα by over-expressing IL-2Rα in representative cell lines. Methods Levels of IL-2Rα were evaluated in the human natural killer cell line NK-92 and the NKTCL cell line SNK-6. Lentiviral vectors were used to express latent membrane protein 1 (LMP1) in NK-92 cells, and IL-2Rα in both NK-92 and SNK-6 cells. The biological effects of these genes on proliferation, apoptosis, cell cycle distribution, and chemosensitivity were analyzed. Results Expression of IL-2Rα was significantly higher in SNK-6 cells than in NK-92 cells. Expressing LMP1 in NK-92 cells remarkably up-regulated IL-2Rα levels, whereas selective inhibitorss of the proteins in the MAPK/NF-κB pathway significantly down-regulated IL-2Rα. IL-2Rα overexpression in SNK-6 cells promoted cell proliferation by altering cell cycle distribution, and induced resistance to gemcitabine, doxorubicin, and asparaginase. These effects were reversed by an anti-IL-2Rα antibody. Conclusions Our results suggest that LMP1 activates the MAPK/NF-κB pathway in NKTCL cells, up-regulating IL-2Rα expression. IL-2Rα overexpression promotes growth and chemoresistance in NKTCL, making this interleukin receptor a potential therapeutic target.

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. It is also characterized by heavy infiltration with non-malignant leucocytes. The EBV-encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signaling pathways which collectively promote cell proliferation and survival, angiogenesis, invasiveness, and aerobic glycolysis. LMP1 also affects cell-cell interactions, antigen presentation, and cytokine and chemokine production. Here, we discuss how LMP1 modulates local immune responses that contribute to the establishment of the NPC tumor microenvironment. We also discuss strategies for targeting the LMP1 protein as a novel therapy for EBV-driven malignancies. [ABSTRACT FROM AUTHOR]

The involvement of the central nervous system (CNS) in Hodgkin lymphoma (HL) has been rarely reported, especially in its primary isolated form. Herein, we present a case of a 33-year-old woman, who received immunosuppressive treatment due to ulcerative colitis (at the beginning azathioprine and sulfasalazine, changed to mesalazine), with repetitive episodes of loss of consciousness for a few weeks and with no other symptoms. Magnetic resonance imaging scans of the head revealed a tumor in the lateral part of the left temporal lobe and in the cerebellum. Moreover, a subsequent computed tomographic scan of the chest revealed diffuse tumorous lesions in the lungs. The brain tumor was resected and a tumorous lesion resected from the lungs was biopsied. The histopathological analysis confirmed the final diagnosis of HL localized in the CNS with concomitant pulmonary lymphomatoid granulomatosis (LYG) grade 1. After the patient underwent radiotherapy and chemotherapy, the patient showed complete regression of lesions in the CNS and lungs, which was confirmed by positron emission tomographic scan. LYG and CNS-HL are rare proliferative disease derived from lymphocytes B and associated with EBV infections. An association between LYG and other autoimmune disorders has been reported, but to the best of our knowledge, this is the first case of the CNS-HL associated with lymphatoid granulomatosis. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) represent a promising treatment option for EBV-associated post-transplantation lymphoproliferative disorders (PTLD). However, production of EBV-CTLs is often complicated and expensive. In the present study, we sought to establish an easy-to-use and economical production protocol for EBV-CTLs. EBV-CTLs were generated using latent membrane protein 1 (LMP1) peptides based on a modified generation protocol of cytokine-induced killer (CIK) cells. After 2-week culture, cells were well expanded (median total cell number: 9.82 × 109; median expansion fold: 107.8) and the median EBV LMP1-specific CD8+ T cell number was 8.94 × 108 (median frequency: 6.7%). However, the EBV-CTL products, unlike CIK cells, did not exhibit NK-like anti-tumor activity. Furthermore, the clinical efficacy of EBV-CTLs was demonstrated with a successful treatment of PTLD on a compassionate use basis in a patient following haploidentical hematopoietic stem cell transplantation. This study indicates the safety and efficacy of EBV LMP1-specific CTLs generated based on a modified generation protocol of CIK cells. Further investigation in a well-designed clinical study is warranted. [ABSTRACT FROM AUTHOR]

Several reports have demonstrated that Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1), which is transferred by extracellular vesicles (EVs) or exosomes, can promote cancer progression. However, its mechanism is still not fully understood. In the present study, we demonstrated that EV packaged LMP1 can activate normal fibroblasts (NFs) into cancer-associated fibroblasts (CAFs). The NF-κB p65 pathway is the key signal that promotes the activation of NFs to CAFs in nasopharyngeal carcinoma (NPC). In activated CAFs, aerobic glycolysis and autophagy were increased. Moreover, glucose uptake and lactate production were decreased, and mitochondrial activity in tumor cells was enhanced, which supported the Reverse Warburg Effect (RWE). During this process, upregulation of MCT4 in CAFs and MCT1 in tumor cells was observed. The NF-κB p65 pathway also plays an important role in the regulation of MCT4. Furthermore, co-culture with CAFs promoted the proliferation, migration and radiation resistance of NPC cells. And EV packaged LMP1 promoted tumor proliferation and pre-metastatic niche formation by activating CAFs in vivo. Our findings indicate that EV packaged LMP1-activated CAFs promote tumor progression via autophagy and stroma-tumor metabolism coupling. [ABSTRACT FROM AUTHOR]

Objective: Regulatory T cells (Tregs) are critical factors that impair antitumor immunity. Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is one of the most pathogenic factors in nasopharyngeal carcinoma (NPC). However, the role of EBV-encoded LMP1 in regulating Treg generation in NPC remains unclear.Materials and Methods: The in vitro stability of activated Tregs (aTregs) influenced by LMP1 was analyzed by flow cytometry. The inhibitory effects of LMP1-HONE1 antigen-induced aTregs on tumor-associated antigen (TAA)-specific T cells were analyzed in vitro and in vivo. Finally, the expression of LMP1, Foxp3, and enhancer of zeste homolog 2 (EZH2) were analyzed in samples from 86 NPC patients by immunohistochemistry and immunofluorescence.Results: LMP1 upregulated the expression of EZH2, which increased the stability of aTregs in vitro. EZH2 inhibitor, DZnep, depleted LMP1-HONE1 antigen-induced aTregs in vitro and led to potent TAA-specific T cell antitumor immunity in vivo. In NPC tissues, LMP1 expression level was positively correlated with the number of EZH2+ Tregs, which was positively correlated with clinical stage and overall survival.Conclusions: EZH2 is essential for maintaining the stability and inhibitory functions of aTregs that are induced by EBV-encoded LMP1 in NPC. [ABSTRACT FROM AUTHOR]

Purpose: Little is known about the clinical significance of CD47 expression and its association with Epstein–Barr virus (EBV) infection in patients with nasopharyngeal carcinoma (NPC). The aim of this study was to clarify the prognostic value and role of CD47 in EBV-associated NPC. Materials and Methods: Sixty-six cases of non-metastatic NPC were retrospectively reviewed. Tissues were collected for immunohistochemical staining of CD47 and the EBV-encoded oncoprotein latent membrane protein 1 (LMP1). Western blotting and quantitative real-time PCR were performed to determine the CD47 and LMP1 levels in common human NPC cell lines. Additionally, CD47 and LMP1 expression in a constructed EBV-positive human NPC cell (CNE-2-EBV+) and a stable cell line transfected with LMP1 plasmid (CNE-2-LMP1) was assessed. Next, we used Western blotting to assess the decrease in CD47 expression on CNE-2-LMP1 cells after transfecting them with small interfering RNA (siRNA)-targeting LMP1. Results: In NPC patients, CD47 overexpression was significantly associated with disease recurrence (P=0.010), leading to poorer disease-free survival (DFS; P=0.002) and overall survival (P=0.021). Multivariate Cox proportional hazards models demonstrated that CD47 (HR=5.452, P=0.016) was an independent prognostic factor of DFS. Moreover, CD47 expression was associated with plasma EBV-DNA copy number and LMP1 tissue expression. Among the human NPC cell lines, CD47 and LMP1 expression was notably higher in the EBV-positive C666-1 cell line than in the EBV-negative cell lines. Furthermore, EBV infection upregulated CD47 expression via LMP1-mediated pathways in human NPC cells. Conclusion: This study indicated that CD47 is related to EBV infection in NPC patients, and it is a feasible biomarker. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is an epithelial tumor with high prevalence in southern China and Southeast Asia. NPC is well associated with the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) 30 bp deletion by having its vital role in increased tumorigenicity and decreased immune recognition of EBV-related tumors. This study developed an InnoPrimers-duplex qPCR for detection of NPC blood circulating LMP1 30 bp deletion genetic biomarker for early diagnosis and treatment response prediction of NPC patients. The analytical and diagnostic evaluation and treatment response prediction were conducted using NPC patients’ whole blood (WB) and tissue samples and non-NPC cancer patients and healthy individuals’ WB samples. The assay was able to detect as low as 20 ag DNA per reaction (equivalent to 173 copies) with high specificity against broad reference microorganisms and archive NPC biopsy tissue and FNA samples. The diagnostic sensitivity and specificity were 83.3% and 100%, respectively. The 30 bp deletion genetic biomarker was found to be a good prognostic biomarker associated with overall clinical outcome of NPC WHO type III patients. This sensitive and specific assay can help clinicians in early diagnosis and treatment response prediction of NPC patients, which will enhance treatment outcome and lead to better life-saving.

Objective: To assess LMP1 gene expression-induced apoptosis in normal nasopharyngeal cells by measuring caspase activity. Material and Methods: LMP1 gene was subcloned into pTracer and pcDNA vector, producing pTracer-LMP1 and pcDNA- LMP1 expression plasmids. The plasmids were then transfected into normal nasopharyngeal cells. LMP1 gene expression-induced apoptosis was accessed by measuring Caspase activities using Caspase-Glo ® 3/7 Assay kit following the manufacturer's protocol. The luminescence intensity was measured by microplate reader. Association of LMP1 gene expression with caspase activation was analysed by independent Sample T test. Results: LMP1 gene expression in normal nasopharyngeal cells is significantly associated with higher caspase activity of apoptosis compare to the vector control with t(-2.142), p value of 0.03. Conclusion: Our results show that, there is association of LMP1 gene expression and caspase activity level in normal nasopharyngeal cell thus support that LMP1 gene expression is involved in apoptosis induction. This in turn suggests that the apoptosis process is potentially involved in the carcinogenesis of nasopharyngeal carcinoma. [ABSTRACT FROM AUTHOR]

Epstein-Barr Virus (EBV) is associated with potentially fatal lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), a serious complication of transplantation. The viral mechanisms underlying the development and maintenance of EBV+ B cell lymphomas remain elusive but represent attractive therapeutic targets. EBV modulates the expression of host microRNAs (miRs), non-coding RNAs that regulate gene expression, to promote survival of EBV+ B cell lymphomas. Here, we examined how the primary oncogene of EBV, latent membrane protein 1 (LMP1), regulates host miRs using an established model of inducible LMP1 signaling. LMP1 derived from the B95.8 lab strain or PTLD induced expression of the oncogene miR-155. However, PTLD variant LMP1 lost the ability to upregulate the tumor suppressor miR-193. Small molecule inhibitors (SMI) of p38 MAPK, NF-κB, and PI3K p110α inhibited upregulation of miR-155 by B95.8 LMP1; no individual SMI significantly reduced upregulation of miR-155 by PTLD variant LMP1. miR-155 was significantly elevated in EBV+ B cell lymphoma cell lines and associated exosomes and inversely correlated with expression of the miR-155 target FOXO3a in cell lines. Finally, LMP1 reduced expression of FOXO3a, which was rescued by a PI3K p110α SMI. Our data indicate that tumor variant LMP1 differentially regulates host B cell miR expression, suggesting viral genotype as an important consideration for the treatment of EBV+ B cell lymphomas. Notably, we demonstrate a novel mechanism in which LMP1 supports the regulation of miR-155 and its target FOXO3a in B cells through activation of PI3K p110α. This mechanism expands on the previously established mechanisms by which LMP1 regulates miR-155 and FOXO3a and may represent both rational therapeutic targets and biomarkers for EBV+ B cell lymphomas.

In the present study, the mechanism by which carboxyl terminal activating region 3 (CTAR3) of latent membrane protein 1 (LMP1), encoded by the Epstein-Barr virus, regulated cell proliferation and protein expression was investigated in the nasopharyngeal epithelial cell line NP69. The deletion mutant LMP1 (LMP1Δ232-351; amino acid residues including 232–351 codons in CTAR3 deleted) was generated by polymerase chain reaction. An NP69-LMP1Δ232-351 cell line was established by retroviral infection. Finally, cell proliferation and protein expression of NP69 cells expressing LMP1Δ232-351 were examined using a cell growth curve and western blot analysis. The results demonstrated: i) The proliferation of NP69-LMP1Δ232-351 cells was significantly decreased compared with cells expressing wild type LMP1 (LMP1WT; n=3; P<0.05); ii) 17 proteins exhibited differential protein expression (>2-fold change) in NP69-LMP1Δ232-351 cells compared with NP69-LMP1WT cells; and iii) LMP1WT was involved in activating the Janus kinase 3 (JAK3) promoter and regulating the expression of JAK3 protein, while LMP1Δ232-351 was almost defective in ability to activate the JAK promoter. These results suggested that LMP1-CTAR3 may be an important functional domain for regulating cell proliferation and protein expression in nasopharyngeal epithelial cells. [ABSTRACT FROM AUTHOR]

Epstein-Barr Virus (EBV) is associated with potentially fatal lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), a serious complication of transplantation. The viral mechanisms underlying the development and maintenance of EBV+ B cell lymphomas remain elusive but represent attractive therapeutic targets. EBV modulates the expression of host microRNAs (miRs), non-coding RNAs that regulate gene expression, to promote survival of EBV+ B cell lymphomas. Here, we examined how the primary oncogene of EBV, latent membrane protein 1 (LMP1), regulates host miRs using an established model of inducible LMP1 signaling. LMP1 derived from the B95.8 lab strain or PTLD induced expression of the oncogene miR-155. However, PTLD variant LMP1 lost the ability to upregulate the tumor suppressor miR-193. Small molecule inhibitors (SMI) of p38 MAPK, NF-κB, and PI3K p110α inhibited upregulation of miR-155 by B95.8 LMP1; no individual SMI significantly reduced upregulation of miR-155 by PTLD variant LMP1. miR-155 was significantly elevated in EBV+ B cell lymphoma cell lines and associated exosomes and inversely correlated with expression of the miR-155 target FOXO3a in cell lines. Finally, LMP1 reduced expression of FOXO3a, which was rescued by a PI3K p110α SMI. Our data indicate that tumor variant LMP1 differentially regulates host B cell miR expression, suggesting viral genotype as an important consideration for the treatment of EBV+ B cell lymphomas. Notably, we demonstrate a novel mechanism in which LMP1 supports the regulation of miR-155 and its target FOXO3a in B cells through activation of PI3K p110α. This mechanism expands on the previously established mechanisms by which LMP1 regulates miR-155 and FOXO3a and may represent both rational therapeutic targets and biomarkers for EBV+ B cell lymphomas. [ABSTRACT FROM AUTHOR]

Nasopharyngeal carcinoma (NPC) is one of the severe head and neck carcinomas, which are rare in western countries but with high incidence in Southern Asia especially South China. NPC is relatively sensitive to radiotherapy, but the prognosis of patients in late stage is poor. The development of NPC is closely related to genetic background, Epstein-Barr virus (EBV) infection and life style. EBV latent membrane protein 1 (LMP1) is an oncoprotein, which plays important roles in the tumorigenesis of NPC. LMP1 was reported to be involved in the regulation of cancer stem cells (CSCs) by immunohistochemistry (IHC) and other surface marker staining methods, but not shown by aldehyde dehydrogenase 1 (ALDH-1) functional assay yet. In this study, we overexpressed LMP1 in two NPC cell lines and found elevated level of cytokeratin 19 (CK19) expression. CK19 is an undifferentiated keratin and a stem cell marker. We also have found an increased number of ALDH positive cells along with LMP1 overexpression in both cell lines. Our data demonstrate that LMP1 regulates the maintenance of ALDH-1 positive NPC cancer stem cells, thus shedding light on target therapy of NPC in clinical application. [ABSTRACT FROM AUTHOR]

Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains; it is associated with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in the cellular trafficking of different proteins, EV cargo sorting, and vesicle formation. We have previously shown that CD63 is important in LMP1 trafficking to EVs, and this also affects LMP1-mediated intracellular signaling including MAPK/ERK, NF-κB, and mTOR activation. Using the BioID method combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as a network of proximal interacting proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism, and transportation. The CD63-only interactome was enriched in Rab GTPases, SNARE proteins, and sorting nexins, while adding LMP1 into the interactome increased the presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of protein enrichment from protein localization and vesicle-mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTOR, Nedd4 L, and PP2A, indicating the formation of a multiprotein complex with CD63, thereby potentially regulating LMP1-dependent mTOR signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into the network of partners required for endocytic trafficking and extracellular vesicle cargo sorting, formation, and secretion.

Abstract Background Natural killer/T-cell lymphoma (NKTCL) is an Epstein–Barr virus (EBV)-associated, highly aggressive lymphoma. Treatment outcome remains sub-optimal, especially for advanced-stage or relapsed diseases. Programmed cell death receptor 1 (PD-1) and PD ligand 1 (PD-L1) have become promising therapeutic targets for various malignancies, but their role in the pathogenesis and their interactions with EBV in NKTCL remains to be investigated. Methods Expression of PD-L1 was measured in NK-92 (EBV-negative) and SNK-6 (EBV-positive) cells by western blot, quantitative real-time PCR and enzyme-linked immunosorbent assay, and flow cytometry, respectively. Latent membrane protein 1 (LMP1)-harboring lentiviral vectors were transfected into NK-92 cells to examine the correlation between LMP1 and PD-L1 expression. Proteins in the downstream pathways of LMP1 signaling were measured in NK-92 cells transfected with LMP1-harboring or negative control vectors as well as in SNK-6 cells. PD-L1 expression on tumor specimens and serum concentration of soluble PD-L1 were collected in a retrospective cohort of patients with Ann Arbor stage I~II NKTCL, and their prognostic significance were analyzed. Results Expression of PD-L1 was significantly higher in SNK-6 cells than in NK-92 cells, at both protein and mRNA levels. Expression of PD-L1 was remarkably upregulated in NK-92 cells transfected with LMP1-harboring lentiviral vectors compared with those transfected with negative control vectors. Proteins in the MAPK/NF-κB pathway were upregulated in LMP1-expressing NK-92 cells compared with the negative control. Selective inhibitors of those proteins induced significant downregulation of PD-L1 expression in LMP1-expressing NK-92 cells as well as in SNK-6 cells. Patients with a high concentration of serum soluble PD-L1 (≥3.4 ng/ml) or with a high percentage of PD-L1 expression in tumor specimens (≥38 %) exhibited significantly lower response rate to treatment and remarkably worse survival, compared with their counterparts. A high concentration of serum soluble PD-L1 and a high percentage of PD-L1 expression in tumor specimens were independent adverse prognostic factors among patients with stage I~II NKTCL. Conclusions PD-L1 expression positively correlated LMP1 expression in NKTCL, which was probably mediated by the MAPK/NF-κB pathway. PD-L1 expression in serum and tumor tissues has significant prognostic value for early-stage NKTCL.

Objective: To evaluate the effect of LMP1 gene expression on oxidative stress generation in normal nasopharyngeal cell. Material and Methods: LMP1 gene were established in pTracer and pcDNA form. The cell then transfection into normal nasopharyngeal cell line. Level of reactive oxygen species (ROS) will be assessed by measuring the fluorescence generated by the chemical 2',7'-Dichlorohydrofluorescein diacetate (DCFH-DA) for pcDNA and cell rox for pTracer. Association of LMP1 gene expression and oxidative stress level were calculate by comparing mean of pTracer and pcDNA with LMP1 gene and without LMP1 gene by use independent Sample T test. Results: There were no association between LMP1 gene expression in both pTracer LMP1 and pcDNA LMP1 compare to vector control cells in normal nasopharyngeal cell. Conclusion: Our results show that, there are no association of LMP1 gene expression and oxidative stress production in normal nasopharyngeal cell thus support that LMP1 gene expression not involve in production of oxidative stress that mediated pathways in the process of nasopharyngeal carcinoma. [ABSTRACT FROM AUTHOR]

Extranodal NK/T-cell lymphoma (ENKTCL) is a highly aggressive mature NK/T-cell neoplasm marked by NK-cell phenotypic expression of CD3ε and CD56. While the disease is reported worldwide, there is a significant geographic variation with its highest incidence in East Asian countries possibly related to the frequent early childhood exposure of Epstein–Barr virus (EBV) and specific ethnic–genetical background, which contributes to the tumorigenesis. Historically, anthracycline-based chemotherapy such as CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone) was used, but resulted in poor outcomes. This is due in part to intrinsic ENKTCL resistance to anthracycline caused by high expression levels of P-glycoprotein. The recent application of combined modality therapy with concurrent or sequential radiation therapy for early stage disease, along with non-anthracycline-based chemotherapy regimens consisting of drugs independent of P-glycoprotein have significantly improved clinical outcomes. Particularly, this neoplasm shows high sensitivity to l-asparaginase as NK-cells lack asparagine synthase activity. Even still, outcomes of patients with advanced stage disease or those with relapsed/recurrent disease are dismal with overall survival of generally a few months. Thus, novel therapies are needed for this population. Clinical activity of targeted antibodies along with antibody–drug conjugates, such as daratumumab (naked anti-CD38 antibody) and brentuximab vedotin (anti-CD30 antibody conjugated with auristatin E), have been reported. Further promising data have been shown with checkpoint inhibitors as high levels of programmed death-ligand 1 expression are observed in ENKTCL due to EBV-driven overexpression of the latent membrane proteins [latent membrane protein 1 (LMP1) and LMP2] with activation of the NF-κB/MAPK pathways. Initial case series with programmed death 1 inhibitors showed an overall response rate of 100% in seven relapsed patients including five with a complete response (CR). Furthermore, cellular immunotherapy with engineered cytotoxic T lymphocytes targeted against LMP1 and LMP2 have shown encouraging results with durable CRs as either maintenance therapy after initial induction chemotherapy or in the relapsed/refractory setting. In this paper, we review this exciting field of novel immunotherapy options against ENKTCL that hopefully will change the treatment paradigm in this deadly disease.

Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which mimics a constitutively active receptor, is required for viral transformation of primary B cells. LMP1 is expressed in EBV-infected germinal center (GC) B cells of immunocompetent individuals, suggesting that it may contribute to persistent EBV infection. In this study, we generated and analyzed mice that expressed LMP1 under the control of the CD19 or activation-induced cytidine deaminase (AID) promoter. Expression of LMP1 induced activation of B cells but severely inhibited their differentiation into antibody-secreting cells (ASCs) in vitro and GC B cells in vivo. LMP1-expressing (LMP1+) B cells not only suppressed the functions of wild-type (WT) B cells in in vitro co-culture, but also blocked differentiation of WT B cells into GC B cells and ASCs in immunized bone marrow chimeric mice. Microarray analysis revealed that the gene encoding indoleamine 2,3-dioxygenase 1 (IDO1), a major enzyme involved in the tryptophan metabolic process, was highly induced by LMP1. Either inhibition of IDO1 activity by methyl- l -tryptophan or knockout of Ido1 in LMP1+B cells could rescue WT B cells from such suppression. IDO1-induced tryptophan consumption and production of tryptophan metabolites appeared to be responsible for inhibition of B-cell function. We conclude that LMP1 expression in antigen-committed B cells not only directly impairs GC B-cell differentiation, but also indirectly inhibits the functions of neighboring B cells, resulting in suppression of humoral immune responses. Such bystander inhibition by LMP1+B cells may contribute to immune evasion by EBV. [ABSTRACT FROM AUTHOR]

Diffuse large B cell lymphoma (DLBCL) has previously been demonstrated to contribute to the mortality of lymphoma with various aggressive features. The prognostic role of the biomarkers latent membrane protein (LMP) 1 and microRNA‑(miR)‑155 in DLBCL remain controversial. The present study primarily aimed to assess the effect of LMP1 and miR‑155 on the survival of DLBCL patients, and additionally evaluate the clinical features to observe their influence on outcomes, compared with previous studies. Formalin‑fixed and paraffin‑embedded samples were collected from our center between May 2010 and December 2011. Microarray analysis, immunohistochemical analysis and reverse transcription‑quantitative polymerase chain reaction were used to evaluate the expression of LMP1 and miR‑155. The association between biomarkers or clinical features and patient outcomes was assessed using the log‑rank statistical test, Cox proportional hazard model and Kaplan‑Meier method. SPSS software was used to statistically analyze the data. A total of 82 patients were included in the present study. The results demonstrated that high expression of LMP1 and miR‑155 may be associated with a poor progression‑free survival rate, while a high International Prognostic Index score and high expression of LMP1 may be associated with a poor overall survival rate. These results indicated that LMP1 and miR‑155 may be novel and reliable biomarkers for the prognostic prediction of lymphoma, and will potentially be analyzed in the future to evaluate patient prognosis. [ABSTRACT FROM AUTHOR]

Objective: To prepare the recombinant lentivirus over-expressing anti -latent membrane protein -1 (anti-LMP-1) single chain variable fragment (scFv) antibody gene, then construct and identify anti-LMP-1 gene modified human peripheral blood mononuclear cells (PBMCs). Methods: The primers of anti-LMP-1 scFvNCD28 and CD3£ were designed according to the GenBank. IgG1-Fc were obtained from existing pVitro-IgG-1 vector. All these sequences mentioned above were amplified and inserted into the lentiviral expression plasmid pCDH to construct protein expression vectors(pCDH-anti-LMP-1-IgG-Fc-CD28-CD3£, hereafter refer to as CAR; control vector pCDH-CD28-CD3£, hereafter refer to as VEC). CAR and VEC lentiviruses were prepared through three-plasmid packaging system (shuttle plasmid pCDH, packaging plasmids pSPAX2 and pMD2.G). The PBMCs were isolated from healthy donors and infected with concentrated pCDH-VEC and pCDH-CAR lentivirus separately. The expression of green fluorescent protein(GFP), as a surrogate reporter of target protein, was detected by fluorescence microscope and flow cytometry. Results: Over-expression vectors, VEC and CAR, were successfully constructed. The concentrated recombinant lentiviruses containing VEC or CAR could infect PBMCs and GFP as a reporter protein could be observed through fluorescence microscope and flow cytometry. The percentage of GFP+ cells among VEC or CAR lentivirus transfected PBMCs were 42.5% and 51%. Conclusion: The anti-LMP-1 gene modified PBMCs were successfully established, which lays an experimental foundation for further research on its cytotoxic effect on LMP-1 positive tumor cells. [ABSTRACT FROM AUTHOR]

Extranodal NK/T-cell lymphoma (ENKTCL) is a highly aggressive mature NK/T-cell neoplasm marked by NK-cell phenotypic expression of CD3ε and CD56. While the disease is reported worldwide, there is a significant geographic variation with its highest incidence in East Asian countries possibly related to the frequent early childhood exposure of Epstein-Barr virus (EBV) and specific ethnic--genetical background, which contributes to the tumorigenesis. Historically, anthracycline-based chemotherapy such as CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone) was used, but resulted in poor outcomes. This is due in part to intrinsic ENKTCL resistance to anthracycline caused by high expression levels of P-glycoprotein. The recent application of combined modality therapy with concurrent or sequential radiation therapy for early stage disease, along with non-anthracycline-based chemotherapy regimens consisting of drugs independent of P-glycoprotein have significantly improved clinical outcomes. Particularly, this neoplasm shows high sensitivity to L-asparaginase as NK-cells lack asparagine synthase activity. Even still, outcomes of patients with advanced stage disease or those with relapsed/recurrent disease are dismal with overall survival of generally a few months. Thus, novel therapies are needed for this population. Clinical activity of targeted antibodies along with antibody--drug conjugates, such as daratumumab (naked anti-CD38 antibody) and brentuximab vedotin (anti-CD30 antibody conjugated with auristatin E), have been reported. Further promising data have been shown with checkpoint inhibitors as high levels of programmed death-ligand 1 expression are observed in ENKTCL due to EBV-driven overexpression of the latent membrane proteins [latent membrane protein 1 (LMP1) and LMP2] with activation of the NF-ΚB/MAPK pathways. Initial case series with programmed death 1 inhibitors showed an overall response rate of 100% in seven relapsed patients including five with a complete response (CR). Furthermore, cellular immunotherapy with engineered cytotoxic T lymphocytes targeted against LMP1 and LMP2 have shown encouraging results with durable CRs as either maintenance therapy after initial induction chemotherapy or in the relapsed/refractory setting. In this paper, we review this exciting field of novel immunotherapy options against ENKTCL that hopefully will change the treatment paradigm in this deadly disease. [ABSTRACT FROM AUTHOR]

Many studies have implicated EBV in the pathogenesis as well as carcinogenesis of a relevant range of malignant squamous cell carcinomas. The present retrospective study was designed to: (1) investigate the frequency of EBV infections associated with the pancreatic carcinoma, (2) explore the impact this virus on the expressed histopathological grades and the stages of those cancers; and (3) screen the expressedimmunohistochemical- EBV Latent Membrane Protein-1 reactions, as referred to signal scores and intensities of these reactions in the tested specimens. The current retrospective study enrolled a number of 29 paraffinized pancreatic tissues from the archives of the period 2011 - 2017 of the major hospitals and many private histopathological laboratories in Baghdad. The research included 19 pancreatic carcinomatous tissues as well as 10 biopsies from an apparently normal pancreatic tissues. Each 4 mm - thick sections of the requested tissue block was stuck on a positive charged slide to be used for an immunohistochemical (IHC) technique for detecting EBV Latent Membrane Protein 1 using monoclonal primary antibodies against them. Histopathological examination revealed that well, moderate and poor grades of pancreatic carcinomas constituted 21%, 63% and 16%, respectively. The EBV- LMP1 was detected in 42% (8 out of 19) of pancreatic cancerous tissues. Seven out of eight tissues with positive EBV-LMP1 reactions have moderate differentiated grade, while only one tissue (1 out of 8) have well differentiated grade and no expression of poor differentiation. It could possibly point that EBV infections in pancreatic carcinomas could have an initiating and/or cofactor roles, in collaboration with other important oncogenic factors or agents in the pancreatic carcinogenesis which could happened as an early- event along other molecular attacks in this process. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

Latent membrane protein 1 (LMP1) is a primary oncogene encoded by the Epstein‐Barr virus, and various portions of LMP1 are detected in nasopharyngeal carcinoma (NPC) tumor cells. LMP1 has been extensively studied since the discovery of its transforming property in 1985. LMP1 promotes cancer cell growth during NPC development and facilitates the interaction of cancer cells with surrounding stromal cells for invasion, angiogenesis, and immune modulation. LMP1 is detected in 100% of pre‐invasive NPC tumors and in approximately 50% of advanced NPC tumors. Moreover, a small population of LMP1‐expressing cells in advanced NPC tumor tissue is proposed to orchestrate NPC tumor tissue maintenance and development through cancer stem cells and progenitor cells. Recent studies suggest that LMP1 activity shifts according to tumor development stage, but it still has a pivotal role during all stages of NPC development. [ABSTRACT FROM AUTHOR]