• BMB Reports
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• 제49권4호
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• pp.226-231
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• 2016

Epstein Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) plays a pivotal in an EBV episome replication and persistence. Despite considerable attempts, there are no EBV drugs or vaccines. We attempted to eradicate EBV episomes by targeting EBNA1 using the transcription activator-like effector nucleases (TALEN) (E1TN). E1TN-mediated transient knockout (KO) of EBNA1 reduced EBNA1 expression, and caused significant loss of EBV genomes and progressive death of EBV-infected cells. Furthermore, when a mixture of EBV-infected Burkitt's lymphoma (BL) cells and EBV-negative BL cells was targeted by E1TN, EBV-negative cells were counter-selected while most EBV-infected cells died, further substantiating that EBNA1 KO caused selective death of EBV-infected cells. TALEN-mediated transient targeting of EBNA1 attenuated the growth of EBV-infected cells, implicating a possible therapeutic application of E1TN for EBV-associated disorders.

• Asian Pacific Journal of Cancer Prevention
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• 제17권7호
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• pp.3411-3416
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• 2016

Background: Nasopharyngeal carcinoma (NPC), a malignancy arising from the epithelial lining of the nasopharynx, is distinct from others cancers in terms of its epidemiologic features. It is rare in most parts of the world except for a few regions with populations of Mongoloid origin. Objectives: To study the expression pattern of Epstein Barr virus (EBV) encoded oncoproteins EBNA1 and LMP1 in different histological types of NPC and to correlate expression patterns with sex, age and histological types. Materials and Methods: A total of 40 formalin-fixed, paraffin-embedded NPC biopsy samples and tissues from 20 healthy controls were collected to study the expression level of EBNA1 and LMP1 using immunohistochemistry. Results: EBNA1 and LMP1 expression was found in 92.5% and 90% respectively, of the cases and none of the control specimens. The expression patterns of EBNA1 and LMP1 were determined to be statistically significant (p<0.05) when correlated with sex, age and histological distributions. Also immunohistochemistry was found to be a sensitive technique in the detection of EBV. Conclusions: The study reveals that the potent oncoproteins EBNA1 and LMP1 were over expressed in our population cohort. Our findings are to some extent inconsistent with earlier reports as our population showed a higher expression of both EBNA1 and LMP1 compared to other studies.

• Molecules and Cells
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• 제21권2호
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• pp.302-307
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• 2006

Two Epstein-Barr virus (EBV) types, type 1 and type 2, maintain the same allelic specificity at four genomic loci encoding the EBNA2, -3A, -3B, and -3C proteins. We have previously described 16 EBV-transformed B-lymphoblastoid cell lines derived from Korean cancer patients, and the EBNA2 types of the EBV isolates therein. In this study, the allelic types of the EBNA2, -3A, -3B, and -3C genes of these EBV isolates were determined. We report the identification of two distinct types of naturally occurring intertypic recombinants, one with genotype EBNA2 type1/EBN3A, -3B, -3C type 2 and the other with genotype EBNA2, -3A type 1/EBNA3B, -3C type 2. The existence of these intertypic recombinants indicates that various intertypic EBV strains may be circulating in the human population, in addition to typical EBV-1 and EBV-2 strains.

• Pediatric Infection and Vaccine
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• 제5권1호
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• pp.121-127
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• 1998

목 적 : 산모 혈청 및 그들의 신생아 제대혈청의 Viral Capsid Antigen에 대한 IgG 항체(VCA IgG)와 Epstein Barr Virus Nuclear Antigen에 대한 IgG 항체(EBNA IgG)의 양성율 및 항체가 수준, 항체의 경태반 전이율에 대해 알아보고자 한다. 방 법 : 1997년 1월 1일부터 5월 30일까지 고려대학교 안산병원에 정상분만을 위해 내원한 산모와 그들의 신생아 제대에서 혈액을 채취하여 효소결합면역흡착검사(Enzyme Linked Immunosorbent Assay) 방법인 ETI-VCA-G Kit와 ETI-EBNA-G Kit (Sorin Biomedica Diagnostics S.p.A, Saluggia, Italy)를 이용하여 VCA IgG와 EBNA IgG를 측정하였다. 항체가는 정해진 방법에 따라 임의 단위(Arbitrary Unit : AU)로 구하였으며 20AU/m1 이상인 경우 양성으로 판정하였다. 신생아 중 재태 연령 37주 미만, 또는 출생 체중 2,500g 미만인 경우는 대상에서 제외하였다. 결 과 : 1) 대상 산모와 신생아는 42쌍(남아 23명, 여아 19명)이었으며 산모 평균 연령 $29.5{\pm}3.0$세, 신생아 재태 연령 $39.9{\pm}1.0$주, 신생아 출생 체중 $3.48{\pm}0.39kg$이었다. 2) VCA IgG 양성률은 산모, 신생아 모두 100%였고 항체 역가는 산모 $260{\pm}201AU/ml$, 신생아 $278{\pm}252AU/ml$였으며 통계학적으로 유의한 차이는 없었다. 산모와 신생아의 VCA IgG 역가 사이에는 유의한 상관관계가 있었으며(r=0.5214, P<0.001) 산모의 역가와 산모 연령, 신생아 제대의 역가와 산모 연령, 재태 연령, 출생 체중 사이에는 유의한 상관관계가 없었다. 3) EBNA IgG 양성율은 산모, 신생아 모두 100%였으며 항체 역가는 산모 $132{\pm}94AU/ml$, 신생아 $149{\pm}104AU/ml$였으며 통계학적으로 유의한 차이는 없었다(P=0.15). 산모와 신생아의 EBNA IgG 역가 사이에는 유의한 상관관계가 있었으나(r=0.7244, P<0.001), 산모의 역가와 산모 연령, 신생아 제대의 역가와 산모 연령, 재태 연령, 출생 체중 사이에는 유의한 상관관계가 없었다. 4) 산모의 VCA IgG와 EBNA IgG 역가 사이에는 유의한 상관관계가 없었다. 결 론 : 산모와 신생아 제대혈의 VCA IgG와 EBNA IgG는 양 군 모두 100%의 양성율을 보였으며, 각각의 역가도 산모와 신생아 모두 비슷한 수준을 보였다.

• BMB Reports
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• 제34권4호
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• pp.322-328
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• 2001

Excision and amplification of pre-determined, large genomic segments (taken directly from the genome of a natural host, which provides an alternative to conventional cloning in foreign vectors and hosts) was explored in human cells. In this approach, we devised a procedure for excising a large segment of human genomic DNA, the iNOS gene, by using the Cre/loxP system of bacteriophage P1 and amplifying the excised circles with the EBNA-1/oriP system of the Epstein-Barr virus. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into the 5'-UTR and 3'-UTR regions of the iNOS gene, together with an oriP sequence for conditional replication. The traps-acting genes cre and EBNA-1, which were under the control of a tetracycline responsive $P_{hcmv^*-1}$ promoter, were also inserted into the 5'-UTR and 3'-UTR regions of the iNOS gene, respectively, by homologous recombination. The strain carrying the inserted elements was stably maintained until the excision and amplification functions were triggered by the induction of cre and EBNA-1. Upon induction by doxycycline, Cre excised the iNOS gene that was flanked by two ZoxP sites and circularized it. The circularized iNOS gene was then amplified by the EBNA-1/oriP-system. With this procedure, approximately a 45.8-kb iNOS genomic fragment of human chromosome 17 was excised and successfully amplified in human cells. Our procedure can be used effectively for the sequencing of unclonable genes, the functional analysis of unknown genes, and gene therapy.

• IMMUNE NETWORK
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• 제11권1호
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• pp.50-58
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• 2011

Background: Epstein-Barr virus associated gastric lymphoepithelioma-like carcinoma (LELC) is characterized by the intensive infiltration of lymphoid cells, the presence of EBV, and the better prognosis over typical adenocarcinoma. Thus, it was assumable that viral latent proteins may be responsible for the recruitment of a certain T cell repertoire to EBV-associated gastric carcinoma. Methods: To examine above possibility, EBV gene expression in gastric carcinoma tissues and usage of TCR among the tumor infiltrating lymphocytes were analyzed. Results: EBV specific DNA and EBERs RNA were detected in 4 out of 30 patients. RT-PCR analysis revealed that all 4 of EBV-positive tumor tissues expressed EBNA1 mRNA and BARTs and LMP2a was detected only one sample out of 4. However, the EBNA2 and LMP-1 transcripts were not detected in these tissues. $CD8^+$ T cells were the predominant population of infiltrating lymphocytes in the EBV-positive gastric carcinoma. According to spectra type analysis of infiltrating T cells, 10 predominant bands were detected by TCR $V{\beta}$ CDR3 specific RT-PCR from 4 EBV-positive tumor tissues. Sequence analysis of these bands revealed oligoclonal expansion of T cells. Conclusion: These findings suggest that clonally expanded T cells in vivo might be a population of cytotoxic T cells reactive to EBV-associated gastric carcinoma.

• Bulletin of the Korean Chemical Society
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• 제33권11호
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• pp.3676-3680
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• 2012

An Epstein-Barr virus (EBV)-based plasmid contains the EBV nuclear antigen 1 (EBNA1) gene and EBV replication origin (oriP) sequence. Since EBNA1 (the only EBV-encoded protein) is combined with oriP, it is replicated simultaneously with chromosomal DNA in human, primate, and canine cells and is faithfully segregated at a stable copy number upon cell division. Consequently, it can be used to stably express gene inserts over a prolonged time in target cells. We have previously shown that the polyamidoamine (PAMAM) dendrimer can be surface-modified with L-arginine. Arginine is present at a high frequency in the transactivator of transcription (Tat) sequences of human immunodeficiency virus (HIV). It presents high membrane permeability and permits effective transfer of DNA inside the cells. In this study, we constructed two kinds of recombinant DNA by inserting the luciferase gene and enhanced green fluorescence protein (eGFP) gene as reporter genes into the pCEP4 plasmid vector. We measured dynamic light scattering (DLS) and zeta potential after preparing PAMAM-based cationic polymer/EBV-based plasmid complexes. We performed transfection of HEK 293 cell lines with the polyplexes, and monitored luciferase activity and green fluorescence protein (GFP) expression. Our results show that PAMAM-based cationic polymer/EBV plasmid complexes provide enhanced and sustained gene expression.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.620-627
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• 2000

To determine the prevalence of genetic polymorphisms in Epstein-Barr virus (EBV) strains in the Korean population, the restriction site polymorphisms for BamHI and XhoI enzymes were analyzed with 16 EBV isolates from cancer patients and 7 EBV isolates from healthy carriers, using polymerase chain reaction techniques. None of the 23 isolates were found to carry an extra BamHI site in the BamHI F-fragment (f-variant). Of the 12 type-1 isolates from the cancer patients, 10 lost both the LMP1 XhoI site and the BamHI site between the BamHi W1* and I1* fragments (a W1*I1* fusion variant or type C). The latter W1*I1* fusion variant was due to a mutation of thymidine to adenine, as evidenced by a sequence analysis. The remaining two type-1 isolates showed either no variation at both sites or the loss of only the XhoI site. In contrast, two type-2 isolates and two intertypic recombinants with a type-1 allele at the EBNA2 locus and type-2 alleles at all or some of the EBNA3 loci retained both enzyme sites. In similar analyses of the 7 isolates from the healthy carriers, five of six type-1 isolates lost these two sites, however, one type-2 isolate did not. These results clearly indicate a strong association of both the LMP1 XhoI site loss and the W1*I1* fusion variant with the type-1 rather than the type-2 EBV strains circulating in the immunocompetent Korean carriers.

• Asian Pacific Journal of Cancer Prevention
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• 제15권5호
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• pp.2001-2006
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• 2014

The objective of this study was to investigate the diagnostic significance of EBV antibody combined detection for nasopharyngeal carcinoma (NPC) in a high incidence region of southern China. Two hundred and eleven untreated NPC patients, 203 non-NPC ENT patients, and 210 healthy controls were recruited for the study. The titers of VCA/IgA and EA/IgA were assessed by immunoenzyme assay, and the levels of Rta/IgG and EBNA1/IgA were determined by enzyme-linked immunosorbent assay. The levels of VCA/IgA, EA/IgA, Rta/IgG and EBNA1/IgA demonstrated no association with gender or age (p>0.05). The receiver operating characteristic curve and the area under the curve were used to evaluate the diagnostic value. The sensitivity of VCA/IgA (98.1%) and the specificity of EA/IgA (98.5%) were the highest. When a logistic regression model was used to combine the results from multiple antibodies to increase the accuracy, the combination of VCA/IgA+Rta/IgG, whose area under the curve was 0.99, had the highest diagnostic efficiency, and its sensitivity, specificity and Youden index were 94.8%, 98.0% and 0.93 respectively. The data suggest that the combination of VCA/IgA+Rta/IgG may be most suitable for NPC serodiagnosis.

• 대한세포병리학회지
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• 제12권2호
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• pp.73-80
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• 2001

The prevalence of Epstein-Barr virus(EBV) in the uterine cervix was investigated to define the possible etiologic role in cervical carcinogenesis. The viral genotyping and LMP-1 30bp deletion were also stooled. The materials included 169 uterine cervical swabs(152 within normal limits, 12 atypical squamous cells of uncertain significance, 3 low grade intraepithelial lesions, and 2 high grade squamous intraepithelial lesion) and 104 uterine cervical tissues obtained from hysterectomy specimens(32 carcinoma in situ, 9 microinvasive squamous cell carcinomas, 37 invasive squamous cell carcinomas, 7 adenocarcinomas, 7 adenosquamous carcinomas, and 12 cervicitis). EBV detected by PCR for EBNA-1 was positive in 52(56.5%) of 92 invasive and noninvasive cervical carcinomas, and 80(48.8%) of 164 inflammatory or normal cervices. The viruses detected in carcinomas were all type A, and LMP-1 30bp deletion form was more frequent in premalignant and malignant cervical lesions than in nonneoplastic cervices. From the above results, it may be concluded that EBV is one of common viruses detected in uterine cervix of Korean women, and type A virus and LMP-1 30bp deletion form may have a role in cervical carcinogenesis.