Latent Membrane Protein 1 Gene Research Articles

Evolutionary analysis of LMP-1 genetic diversity in EBV-associated nasopharyngeal carcinoma: Bioinformatic insights into oncogenic potential

EBV latent membrane protein 1 (LMP-1) is an important oncogene involved in the induction and maintenance of EBV infection and the activation of several cell survival and proliferative pathways. The genetic diversity of LMP-1 has an important role in immunogenicity and tumorigenicity allowing escape from host cell immunity and more metastatic potential of LMP-1 variants. This study explored the evolutionary of LMP-1 in EBV-infected patients at an advanced stage of nasopharyngeal carcinoma (NPC). Detection of genetic variability in LMP-1 genes was carried out using Sanger sequencing. Bioinformatic analysis was conducted for translation and nucleotide alignment. Phylogenetic analysis was used to construct a Bayesian tree for a deeper understanding of the genetic relationships, evolutionary connections, and variations between sequences. Genetic characterization of LMP-1 in NPC patients revealed the detection of polymorphism in LMP-1 Sequences. Motifs were identified within three critical LMP-1 domains, such as PQQAT within CTAR1 and YYD within CTAR2. The presence of the JACK3 region at specific sites within CTAR3, as well as repeat regions at positions (122−132) and (133–143) within CTAR3, was also annotated. Additionally, several mutations were detected including 30 and 69 bp deletions, 33 bp repeats, and 15 bp insertion. Although LMP-1 strains appear to be genetically diverse, they are closely related to 3 reference strains: prototype B95.8, Med- 30 bp deletion, and Med + 30 bp deletion. In our study, one of the strains harboring the 30 bp deletion had both bone and bone marrow metastasis which could be attributed to the fact that LMP-1 is involved in tumor metastasis, evasion and migration of NPC cells. This study provided valuable insights into genetic variability in LMP-1 sequences of EBV in NPC patients. Further functional studies would provide a more comprehensive understanding of the molecular characteristics, epidemiology, and clinical implications of LMP-1 polymorphisms in EBV-related malignancies.

Epstein-Barr virus infection as potential indicator of the occurrence and clinical presentation of systemic lupus erythematosus.

The relationship between Systemic lupus erythematosus (SLE) and Epstein-Barr virus (EBV) infection has been suggested for decades, but the underlying mechanism of the EBV influence on SLE development remains to be elucidated. The goals of this research, which included 103 SLE patients and 99 controls, were to investigate the association of the parameters of EBV infection and SLE, to explore whether pooled demographic, clinical and EBV markers achieve a more significant effect on SLE development than each of them individually, and to evaluate EBV nuclear antigen 1 (EBNA1) and latent membrane protein 1 (LMP1) gene polymorphisms in isolates from SLE patients. Comprehensive results related to serological, molecular and sequence markers of EBV infection in SLE patients demonstrated even 24 times higher possibility of having SLE if there is the presence of anti-EBV-EA(D) (early antigen) IgG antibodies (OR=24.086 95%CI OR=2.86-216.07, p=0.004). There was the same distribution of glucocorticoids (p=0.130), antimalarials (p=0.213), and immunosuppressives (p=0.712) in anti-EBV-EA(D) IgG positive and negative SLE patients. Further, higher anti-EBV-EA(D) IgG antibodies titers were identified as independent factors associated with lymphopenia, hematological SLE manifestation (OR=1.041, 95%CI OR=1.01-1.08, p=0.025, while a higher titer of anti-CA (viral capsid antigen) IgG antibodies (OR=1.015, 95%CI OR=1.01-1.03, p=0.019) and positive RF (rheumatoid factors) (OR=4.871, 95%CI OR=1.52-15.61, p=0.008) were identified as independent factors associated with alopecia within SLE. Finally, novel data on EBV EBNA1 and LMP1 gene polymorphisms in lupus are reported. The results support further investigation targeting EBV as a prognostic marker and therapeutic goal for lupus.

Vaccination against Epstein-Barr Latent Membrane Protein 1 Protects against an Epstein-Barr Virus-Associated B Cell Model of Lymphoma.

In this study, we demonstrate that expression of viral latent membrane protein 1 (LMP1) in a mouse B cell line renders the animals responsive to protection from a 38C13-LMP1 tumor challenge with a novel vaccine. The Epstein-Barr virus (EBV) preferentially infects circulating B lymphocytes, has oncogenic potential, and is associated with a wide variety of B cell lymphomas. EBV is ectotrophic to human cells, and currently there are no B cell animal models of EBV-associated lymphoma that can be used to investigate vaccine immunotherapy. Since most EBV-infected human tumor cells express latent membrane protein 1 (LMP1) on their surface, this viral antigen was tested as a potential target for an anticancer vaccine in a mouse model. Here, we describe a new mouse model of LMP1-expressing B cell lymphoma produced with plasmid transduction of 38C13 into mouse B cells. The expression of LMP-1 was confirmed with a western blot analysis and immunocytochemistry. We then designed a novel LMP1 vaccine, by fusing viral antigen LMP1 surface loop epitopes to the surface of a viral antigen carrier, the Tobacco Mosaic virus (TMV). Vaccinated mice produced high titer antibodies against the TMV-LMP1 vaccine; however, cellular responses were at the baseline, as measured with IFNγ ELISpot. Despite this, the vaccine showed significant protection from a 38C13-LMP1 tumor challenge. To provide additional immune targets, we compared TMV-LMP1 peptide immunization with DNA immunization with the full-length LMP1 gene. Anti-LMP1 antibodies were significantly higher in TMV-LMP1-vaccinated mice compared to the DNA-immunized mice, but, as predicted, DNA-vaccinated mice had improved cellular responses using IFNγ ELISpot. Surprisingly, the TMV-LMP1 vaccine provided protection from a 38C13-LMP1 tumor challenge, while the DNA vaccine did not. Thus, we demonstrated that LMP1 expression in a mouse B cell line is responsive to antibody immunotherapy that may be applied to EBV-associated disease.

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 30bp 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.

Study of Three Potential Diagnostic Biomarkers in Nasopharyngeal Carcinoma Samples from Guilan, North of Iran.

Introduction Finding biomarkers for highly lethal cancers is a priority. Objective The current study was designed to understand the clinical significance of vascular endothelial growth factor (VEGF), latent membrane protein 1 (LMP1), and tumor necrosis factor-α (TNF-α) expression as the biomarkers, and evaluate their correlation with each other, in nasopharyngeal carcinoma (NPC) in the province of Guilan, North of Iran. Methods Gene expression was evaluated in 25 formalin-fixed paraffin-embedded (FFPE) blocks from cases of confirmed NPC and 20 FFPE samples of non-NPC by quantifying messenger ribonucleic acid (mRNA) and protein levels, using real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) methods, respectively. Furthermore, the correlations among the protein levels of different genes, along with the patients' demographic characteristics were assessed. Results Our findings on mRNA and protein levels demonstrated that the expression of the LMP1 gene in the NPC group was significantly elevated compared with that of the non-NPC group. In addition, the protein levels in the NPC group indicated a positive and significant correlation between LMP1 and VEGF expression. It was noted that both protein and mRNA levels showed no significant differences in the expression of TNF-α and VEGF genes between the NPC and control groups. Furthermore, there was no significant relationship between the expression of these proteins and the demographic characteristics of NPC patients. Conclusion Overall, a significant increase in LMP1 expression was observed in NPC patients, which may serve as a diagnostic biomarker for NPC. Also, LMP1 might be involved in NPC progression by inducing VEGF gene expression.

Sequence variations of Epstein-Barr virus LMP1 gene in gastric cancer and chronic gastritis isolates from Iranian patients.

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 C-terminal domain. Most LMP1-mediated signal transduction events are moderated by some functional parts of the cytoplasmic C-terminal 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.ResultsFour 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.

Expression Mechanism of miR-10b in Cervical Cancer Under Treatment of Methoxy-Polyethylene Glycol-Polylactic Drug-Loaded Nanoparticles

This work aimed to analyze expression mechanism of micro ribonucleic acid (RNA)-10b (miR-10b) in cervical cancer (CC) based on Curcumin-monomethoxy polyethylene glycol-polylactic acid-drug-loaded nanoparticles (CUR-MPEG-PLA DLNPs). 36 Kunming mice were selected and randomly rolled into group A and group B, and 18 nude mice were deemed as controls. CUR-MPEG-PLA DLNPs were prepared for the treatment of 18 Kunming mice in group A. The CC tumor tissues of 36 Kunming mice were harvested, the miR-10b of CC cells was extracted, and the expression level of CC cells was detected by fluorescence quantitative polymerase chain reaction (qPCR). The results showed that the average particle size of CUR-MPEG-PLA DLNPs was 362.52±6.34 nm. The tumor inhibition rate of Kunming mice in group A against CC tumor strains 63.35±3.62% was considerably superior to those in group B (26.31 ±2.54%) (P < 0.05). In group A, expression rate of miR-10b (77.78%) was remarkably superior to controls (55.55%) (x2 = 2.364, P < 0.05). In 20 cases of CC tissues with positive expression of latent membrane protein 1 (LMP-1), expression of miR-10b in metastasis group was obviously higher relative to non-metastasis group (Z = −3.214, P < 0.05). In 16 cases of CC tissues with negative expression of LMP-1, expression of miR-10b in metastasis group was also considerably higher versus non-metastasis group (Z = −2.245, P < 0.05). In conclusion, the average particle size of CUR-MPEG-PLA DLNPs was 362.52 ±6.34 nm, the electromotive force was −9.23 mV, the dispersion coefficient was 0.064, the drug loading rate of nanoparticles was 8.54%, and the encapsulation rate was 72.36%. miR-10b has certain correlation with the metastasis of cervical cancer. LMP-1 gene has the function of promoting the metastasis of cervical cancer tumor cells, which may be dependent on miR-10b. CUR-MPEG-PLA DLNPs loading system can inhibit tumor growth obviously, and has excellent therapeutic effect in CC disease.

Epstein–Barr virus LMP1 oncogene polymorphism in tatar and slavic populations in Russian Federation impacting on some malignant tumours

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.

Detection of Epstein-Barr Virus in Saliva and Gen LMP1 among HIV- Infected Patients

Epstein-Barr virus (EBV) is also called human herpes virus 4 (HHV-4), has detected 95% of the population and shows an asymptomatic state. EBV is etiological agent of oral hairy leukoplakia (OHL) in HIV patients. Latent membrane protein 1 ( LMP1 ), an integral EBV protein can modulate growth, differentiation, induce the expression of several cells, activation of antigens, and adhesion molecules. The LMP1 gene has been associated with OHL. Objectives : to determine the prevalence of EBV in saliva and the LMP1 gene in HIV/AIDS patients with EBV positive. Methods : A cross-sectional was conducted on HIV/AIDS patients. The presence of EBV in saliva was done by mciroarray PCR. LMP1 is examined by using nested PCR. Results : The research subjects involved 30 HIV/AIDS patients consisting 70% men and 30% women, with 50 % age group of 31-40 years old and 40% had CD4 counts <200 cells/mm 3 (40%). EBV in saliva was found in 26 out of 30 (87%) HIV patients and LMP1 was detected in 17 patients (65.38%). Conclusion : The high prevalence of EBV in saliva and the LMP1 gene may increase the risk of OHL. Early screening for EBV infection in patients with HIV/AIDS is important to reduce the risk of EBV-associated diseases.

Detection of Epstein-Barr Virus in Saliva and Gen LMP1 among HIVInfected Patients HIVInfected Patients

Epstein-Barr virus (EBV) is also called human herpes virus 4 (HHV-4), has detected 95% of the population and shows an asymptomatic state. EBV is etiological agent of oral hairy leukoplakia (OHL) in HIV patients. Latent membrane protein 1 (LMP1), an integral EBV protein can modulate growth, differentiation, induce the expression of several cells, activation of antigens, and adhesion molecules. The LMP1 gene has been associated with OHL. Objectives: to determine the prevalence of EBV in saliva and the LMP1 gene in HIV/AIDS patients with EBV positive. Methods: A cross-sectional was conducted on HIV/AIDS patients. The presence of EBV in saliva was done by mciroarray PCR. LMP1 is examined by using nested PCR. Results: The research subjects involved 30 HIV/AIDS patients consisting 70% men and 30% women, with 50 % age group of 31-40 years old and 40% had CD4 counts <200 cells/mm3 (40%). EBV in saliva was found in 26 out of 30 (87%) HIV patients and LMP1 was detected in 17 patients (65.38%). Conclusion: The high prevalence of EBV in saliva and the LMP1 gene may increase the risk of OHL. Early screening for EBV infection in patients with HIV/AIDS is important to reduce the risk of EBV-associated diseases.

Tần suất và đột biến gen LMP1 của virus Epstein-Barr ở mẫu sinh thiết vòm của bệnh nhân ung thư vòm mũi họng điều trị tại Bệnh viện Ung bướu Cần Thơ

Latent Membrane Protein 1 (LMP1) gen of Epstein-Barr virus (EBV) (LMP1 EBV) and the mutation in LMP1 EBV were important for the formation of malignant tumors in nasopharyngeal carcinoma (NPC) in patients with EBV infection. The objective of the study was to determine the frequency and loss 30 bp mutation of the LMP1 gene in the biopsy specimen of the NPC patient. A cross sectional descriptive study was carried out of 65 biopsy specimes of patients, who was confirmed as nasopharynreal carcinoma at the Can Tho Oncology Hospital. Polymerase Chain Reaction (PCR) with LMP1 primer (168373-168174) was used to determine the frequency of LMP1 EBV gene and sequening technique was applied to identify LMP1 gene mutation. As a result, the rate of LMP1 EBV in nasopharyx biopsy specimens was 61.5% (40/65). Besides, there was 57.5% (23/40) of 30 bp loss mutant on LMP1 EBV gene was indentified among of LMP1 EBV in nasopharyx biopsy specimens. In conclusions, the rate of LMP1 EBV on biopsy specimens of NPC in Can Tho was 61.5% with the common mutation pattern of 30bp loss.

Characterization of CSF2A fusion gene and its effect on Epstein-Barr virus-positive tumor cells.

We build the latent membrane protein gene latent membrane protein 2A (LMP2A) and the granulocyte-macrophase colony-stimulating factor (GM-CSF) gene fusion gene (CSF2A) and discuss how the CSF2A fusion protein influenced the proliferation and apoptosis of Epstein-Barr virus-positive (EBV+ ) tumor cells. Reverse-transcription polymerase chain reaction (RT-PCR) method was used to amplify the LMP2A gene and GM-CSF gene fragments, respectively, according to the principle of overlap extension in the coding (Gly4Ser)3 polypeptide gene fragments of DNA restructured under the connection. The CSF2A gene could be connected with the pIRES2-enhanced green fluorescent protein vector by recombinant DNA technology and identified by enzyme electrophoresis analysis and DNA sequencing. Then, the recombinant vector was transfected into dendritic cells (DCs); RT-PCR and Western blot analysis were used for testing the CSF2A gene messenger RNA and protein expression. The impacts of CSF2A on the proliferation and apoptosis of EBV+ tumor cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Hochest 33342 staining. We successfully obtained the recombinant vector named pIRES2-CSF2A. The expression of CSF2A could be detected by transfecting pIRES2-CSF2A into DCs. The DCs were cocultured with T lymphocytes and then acted on the EBV+ CNE2 nasopharyngeal carcinoma cells. MTT assay showed that the inhibiting effect of CSF2A obviously increased and the time dependency (**P < 0.01, *P < 0.05) also existed. Hochest 33342 staining showedapoptosis morphological changes of cells in nucleus staining and generated the apoptotic body. Apoptosis cells of the pIRES2-CSF2A group increased significantly at 48 hours. The results showed that the pIRES2-CSF2A recombinant vector was effectively transfected into DCs and the fusion gene CSF2A could promote EBV+ CNE2 cell apoptosis, laying the foundation for the specificity of EBV+ tumor targeting immune gene therapy in the future.

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

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

EPSTEIN–BARR VIRUS IN THE POPULATION OF TWO GEOGRAPHICALLY DIFFERENT REGIONS OF RUSSIA

It is well known that the Epstein–Barr virus (EBV) being widely spread in the human population is also the etiologic agent for a number of malignancies. A notable feature of tumors associated with EBV is their different incidence in various geographical regions, that, as suggested, related with mutational events in multiple loci of the EBV genome and its oncogene, the latent membrane protein 1 (LMP1), associated with the transforming potential of the virus. Given the multi-ethnic composition of Russian population and the diversity of geographical areas and conditions of their residence, it was relevant to examine the representatives of different geographical regions for the nature of their relationship with EBV. To solve this task the antibody response to locally circulating EBV strains, determined by indirect immunofluorescence, was studied in residents of the Central, North Caucasus and Far Eastern Federal Districts, represented by healthy individuals and patients with various head and neck tumors. The levels of antibody titers obtained were compared with the incidence rates of nasopharyngeal tumors (NPT) in population of above Districts. In order to determine possible structural modifications in LMP1 gene of EBV strains persisting in selected geographic regions, samples of the gene have been amplified from a biological material collected by “nested” PCR and sequenced. The results obtained have shown that levels of antibody response to EBV among representatives of the regions included in the study vary significantly. It was found that in residents of the Dagestan and the Chechen Republics, the inhabitants of the North Caucasus Federal District, the correlation between enhanced humoral response to EBV and increased incidence of NPT was detected. Since among NPT the EBV-associated form of nasopharyngeal carcinoma (NPCEBV) is dominated, the findings allow us to suggest that the population of these Republics have genetic predisposition to increased EBV replication and, consequently, an elevated incidence of NPCEBV. The study also showed that representatives of the regions tested are infected with EBV strains, LMP1 variants of which such as B95.8, China 1, Med+, Med– and NC, are known in the literature. However, LMP1 variant, specifically associated with NPCEBV, has not been identified. This study showed for the first time the genetic heterogeneity of EBV strains circulating among population of different geographical regions of the country, and the existence of correlation between antibody response to EBV and the incidence of nasopharyngeal tumors, including NPCEBV.

Higher methylation intensity induced by EBV LMP1 via NF-κB/DNMT3b signaling contributes to silencing of PTEN gene.

Phosphatase and tensin homolog (PTEN) is a major tumor suppressor and usually silenced via the deletion, insertion and mutation. We previously discovered its inactivation via aberrant CpG island methylation. Here, we provide further evidence that EBV latent membrane protein 1(LMP1) can induce a higher intensity of DNA methylation at PTEN CpG islands, inactivating PTEN at the cellular and molecular level. Initially, increased methylation intensity of PTEN CpG islands was observed in EBV-infected nasopharyngeal carcinoma (NPC) cells, accompanied by decreased PTEN expression. In NPC tissue samples showing the methylation at PTEN promoter, LMP1 was highly expressed in higher methylation intensity group relative to lower intensity group, and DNA methyltransferase 3b (DNMT3b) expression was positively correlated with LMP1 expression. Moreover, transfection of LMP1 gene into EBV-negative NPC cells demonstrated that LMP1 up-regulated DNMT3b expression, leading to a higher intensity of PTEN CpG island methylation. Mechanistically, computational prediction and luciferase reporter assay identified a functional NF-κB binding site on DNMT3b promoter and the mutated NF-κB binding site abolished LMP1-mediated DNMT3b activation. Chromatin immunoprecipitation displayed that NF-κB p65 subunit constitutively bound to DNMT3b promoter, supporting the activation of DNMT3b by EBV LMP1 via NF-κB signaling. Furthermore, the expression level of DNMT3b was observed to be increased in the nuclei of LMP1-expressing NPC cells, and a NF-κB inhibitor, PDTC, counteracted LMP1-mediated DNMT3b overexpression. Thus, this study first reports that LMP1-mediated NF-κB can up-regulate DNMT3b transcription, thereby leading to relatively higher methylation intensity at PTEN CpG islands, and ultimately silencing major tumor suppressor PTEN.

Clonal deleted latent membrane protein 1 variants of Epstein-Barr virus are predominant in European extranodal NK/T lymphomas and disappear during successful treatment.

Extranodal natural killer/T-cell lymphomas (NK/TL), rare in Europe, are Epstein-Barr virus (EBV) associated lymphomas with poor outcomes. Here, we determined the virus type and analyzed the EBV latent membrane protein-1 (LMP1) gene sequence in NK/TL from French patients. Six clones of viral LMP1 were sequenced by Sanger technology in blood from 13 patients before treatment with an l-asparaginase based regimen and, for 8 of them, throughout the treatment. Blood LMP1 sequences from 21 patients without any known malignancy were tested as controls. EBV Type A was identified for 11/13 patients and for all controls. Before treatment, a clonal LMP1 gene containing a 30 bp deletion (del30) was found in 46.1% of NK/TL and only in 4.8% of controls. Treatment was less effective in these patients who died more rapidly than the others. Patients with a deleted strain evolving toward a wild-type strain during treatment reached complete remission. The LMP1 gene was sequenced by highly sensitive next-generation sequencing technology in five NK/TL nasopharyngeal biopsies, two of them originating from the previous patients. Del30 was present in 100% of the biopsies; two viruses at least coexisted in three biopsies. These results suggest that del30 may be associated with poor prognosis NK/TL and that strain evolution could be used as a potential marker to monitor treatment.

Characterization of the Variability of Epstein-Barr Virus Genes in Nasopharyngeal Biopsies: Potential Predictors for Carcinoma Progression.

Epstein-Barr virus (EBV) infection is a significant factor in the pathogenesis of nasopharyngeal carcinoma, especially in the undifferentiated carcinoma of nasopharyngeal type (UCNT, World Health Organization type III), which is the dominant histopathological type in high-risk areas. The major EBV oncogene is latent membrane protein 1 (LMP1). LMP1 gene shows variability with different tumorigenic and immunogenic potentials. EBV nuclear antigen 1 (EBNA1) regulates progression of EBV-related tumors; however, the influence of EBNA1 sequence variability on tumor pathogenesis is controversial. The aims of this study were to characterize polymorphisms of EBV genes in non-endemic nasopharyngeal carcinoma biopsies and to investigate potential sequence patterns that correlate with the clinical presentation of nasopharyngeal carcinoma. In total, 116 tumor biopsies of undifferentiated carcinoma of nasopharyngeal type (UCNT), collected from 2008 to 2014, were evaluated in this study. The genes EBNA2, LMP1, and EBNA1 were amplified using nested-PCR. EBNA2 genotyping was performed by visualization of PCR products using gel electrophoresis. Investigation of LMP1 and EBNA1 included sequence, phylogenetic, and statistical analyses. The presence of EBV DNA was significantly distributed between TNM stages. LMP1 variability showed six variants, with the detection of the first China1 and North Carolina variants in European nasopharyngeal carcinoma biopsies. Newly discovered variants Srb1 and Srb2 were UCNT-specific LMP1 polymorphisms. The B95-8 and North Carolina variants are possible predictors for favorable TNM stages. In contrast, deletions in LMP1 are possible risk factors for the most disfavorable TNM stage, independent of EBNA2 or EBNA1 variability. A newly discovered EBNA1 subvariant, P-thr-sv-5, could be a potential diagnostic marker, as it represented a UCNT-specific EBNA1 subvariant. A particular combination of EBNA2, LMP1, and EBNA1 polymorphisms, type 1/Med/P-thr was identified as a possible risk factor for TNM stage IVB or progression to the N3 stage.

Expression of LMP and EBNA genes in Epstein-Barr virus-associated lymphomas in Hu-PBL/SCID mice.

Transplantation of peripheral blood lymphocytes (PBLs) from healthy humans with latent Epstein-Barr virus (EBV) infection into severe combined immunodeficiency (SCID) mice results in development of EBV-associated human B-cell lymphoma. However, the expression of EBV genes in relation to lymphoma development has not been reported. We investigated latent membrane protein (LMP) and EBV nuclear antigen (EBNA) gene expression in PBLs from EBV-positive blood donors and induced-lymphoma cells from SCID mice to elucidate the functions and effects of the EBV genome in the occurrence and development of lymphoma. PBLs were isolated from 9 healthy blood donors and transplanted into SCID mice. Gene expression levels of LMP-1, LMP-2A, and LMP-2B and EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP were monitored by real-time quantitative-polymerase chain reaction (qRT-PCR) in cells from nine EBV-induced lymphomas and in matched lymphocytes from healthy subjects. LMP-1, EBNA-1 and EBNA-2 protein levels were detected by western blotting. As a result, LMP-1, LMP-2A and LMP-2B mRNA levels were upregulated 256-, 38- and 331-fold, respectively, in the EBV-induced lymphoma cells compared with the controls, while EBNA-1 and EBNA-3A mRNA levels were upregulated 1157- and 1154-fold, respectively. EBNA-2, EBNA-3B, EBNA-3C and EBNA-LP mRNAs were detected in lymphoma cells, but not in lymphocytes from EBV-positive blood donors. LMP-1 and EBNA-2 proteins were not expressed in lymphocytes from EBV-positive blood donors, according to western blotting. Weak EBNA-1 expression was observed in lymphocytes from blood donors with latent EBV infection, while LMP-1, EBNA-1 and EBNA-2 protein levels were significantly upregulated in EBV-induced lymphoma cells, consistent with mRNA expression levels detected by qRT-PCR. In conclusion, LMP-1, LMP-2A, LMP-2B, EBNA-1 and EBNA-3A were upregulated in EBV-induced lymphoma cells, while EBNA-2, EBNA-3B, EBNA-3C and EBNA-LP were absent in lymphocytes from humans with latent EBV infection, but were positively expressed in EBV-induced lymphoma cells.

The Epstein-Barr virus latent membrane protein-1 (LMP1) 30-bp deletion and XhoI-polymorphism in nasopharyngeal carcinoma: a meta-analysis of observational studies.

BackgroundEpstein-Barr virus (EBV) is considered to be closely associated with nasopharyngeal carcinoma (NPC), in which EBV-encoded latent membrane protein 1 (LMP1) was found to have an oncogenic role. However, the results published on the LMP1 polymorphism are inconsistent. In the present study, we performed a meta-analysis to determine the frequency of the associations and a more precise association between NPC and EBV LMP1 gene variants (30-bp deletion (del)/XhoI-loss).MethodsEligible articles met the inclusion/exclusion criteria and were identified in the following electronic databases: PubMed, ScienceDirect, and SciELO. Consequently, the data of interest were extracted and plotted in a table to calculate the frequency and odds ratio (OR) of the outcomes of interest (30-bp del-LMP1/XhoI-loss) in patients with NPC. Study quality (Newcastle-Ottawa Scale (NOS)), publication bias, and heterogeneity were assessed.ResultsThirty-one observational studies were included with a total of 2,846 individuals (NPC, n = 1,855; control, n = 991). The risk of bias in relation to study quality evaluated by NOS was considered low. The pooled estimate of the frequency of 30-bp del-LMP1 and XhoI-loss in patients with NPC was 77% (95% confidence interval (CI): 72 to 82) and 82% (95% CI: 71 to 92), respectively. There was an association between 30-bp del-LMP1 and NPC susceptibility (OR = 2.86, 95% CI: 1.35 to 6.07, P = 0.00). Similarly, there was an association between XhoI-loss and NPC (OR = 8.5, 95% CI: 1.7 to 41, P = 0.00). However, when we analyze the co-existence of the 30-bp del-LMP1 and XhoI-loss in patients with NPC, there was no association (OR = 1.09, 95% CI: 0.06 to 18.79, P = 0.002).ConclusionsOur results suggest an association between the 30-bp del-LMP1 and XhoI-loss with NPC susceptibility. However, our data should be interpreted with caution because the sample size was small, and there was heterogeneity between the studies. Thus, future studies are needed with adjusted estimates to simultaneously evaluate multiple factors involved in the development of NPC.Systematic review registrationPROSPERO CRD42014013496.Electronic supplementary materialThe online version of this article (doi:10.1186/s13643-015-0037-z) contains supplementary material, which is available to authorized users.

Expression of latent membrane proteins in Epstein‑Barr virus-transformed lymphocytes invitro.

Infection with Epstein-Barr virus (EBV) induces activation and proliferation of Blymphocytes. Detection of latent membrane protein (LMP)-1 is used to identify the proliferative ability of B cells. However, changes in the expression levels of the three LMPs during EBV-induced Blymphocyte transformation, have not yet been reported. In the present study, the expression levels of LMP-1, LMP-2A and LMP-2B were compared between EBV-transformed Blymphocytes and paired normal lymphocytes. Seven lymphoblast cell lines were established by EBV infection of normal human lymphocytes invitro. The expression levels of LMP genes and LMP-1 protein were determined using quantitative (q)PCR and western blotting in lymphoblasts and normal lymphocytes, respectively. The expression of LMP1, LMP-2A and LMP-2B genes was significantly upregulated in EBV-induced lymphoblasts compared with the normal lymphocytes. The LMP-1 protein level was also significantly increased in EBV-transformed B lymphocytes. Expression of LMP1, LMP-2A and LMP-2B genes was significantly upregulated in EBV-induced lymphoblasts, suggesting LMP genes are important in the transformation of human lymphocytes.