B 세포에서 Epstein-Barr virus microRNA들의 전사 및 성숙

김도년,오상택,이재면,이원근,이숙경,Kim Do Nyun,Oh Sang Taek,Lee Jae Myun,Lee Won-Keun,Lee Suk Kyeong 한국생명과학회 2005 생명과학회지 Vol.15 No.6

우리는 바이러스로는 최초로 miRNA를 생성한다고 보고된 Epstein-Barr virus (EBV)를 대상으로 EBV miRNA biogenesis를 연구했다. 먼저 EVB에 감염된 B 세포인 B95-8로부터 보고된 5 종의 EBV miRNA들인 BHRF1-1, BHRF1-2, BHRF1-3, BART1, 및 BART2 모두가 발현됨을 확인하였다. 그 다음 성숙된 EBV miRNA 서열로부터 예측되는 pri- 와 pre-miRNA들이 B95-8에서 각각 검출되어 EBV miRNA들도 이미 알려진 동물세포 miRNA와 유사한 생성 및 성숙과정을 거칠 가능성을 확인하였다 게놈 상에 2~7개씩 밀집하여 존재하는 동물세포 miRNA들과 유사하게 EBV 게놈의 2부 위에 밀집해서 존재하는 EBV miRNA들도 polycistronic하게 발현되는지 조사한 결과 B95-8에서 BHRF1-1, BHRF1-2 및 BHRF1-3를 포함하는 1,602 뉴클레오타이드의 긴 전사체가 RT-PCR로 확인되었다. 반면 BART1과 BART2는 mono cistronic하게 전사될 가능성이 확인되었다. 본 연구를 통하여 EBV miRNA들도 동물세포 miRNA들과 유사하게 poly cistronic 하게 발현될 수 있으며 pri-와 pre-miRNA 과정을 거쳐 성숙된 miRNA로 생성될 가능성을 확인하였다. We investigated microRNA (miRNA) biogenesis of Epstein-Barr virus (EBV) which is the first virus shown to produce viral miRNAs. As expected, expression of all the reported EBV miRNAs were detected by Northen blot in an EBV-infected B cell line, B95-8; BHRF1-1, BHIU1-2, BHRF1-3, BART1, and BART2. The putative EBV pri-miRWAs and pre-miRNAs predicted from the known mature EBV miRNA sequences were detected by RT-PCR in B95-8 cells. Many animal miRNA genes exist as clusters of 2-7 genes and they are expressed polycistronically. As the EBV miRNAs are clustered in two regions of the EBV genome, we examined whether these clustered EBV miRNA genes are also expressed polycistronically. A long polycistronic transcript with the expected size (1602 bp) corresponding to the BHRF1-1~BHRF1-2~BHRF1-3 was amplified. However, any polycistronic transcript containing both BART1 and BART2 was detectable in B95-8. These results suggest that EBV miRNAs may be processed in a similar way with animal miRNAs and that some of the clustered EBV miRNAs can be transcribed polycistronically.

Significance of Epstein-Barr Virus DNA Quantitation in Donors of Hematopoietic Stem Cell Transplantation

정승원,임지향,조병식,채효진,김명신,김용구,한경자,이종욱,민우성 대한진단검사의학회 2010 Annals of Laboratory Medicine Vol.30 No.6

Background : Epstein-Barr virus (EBV) is a well-known causative agent of various diseases including post-transplant lymphoproliferative disorders. Although the level of EBV viral load in donors is expected to have a direct effect on recipients after hematopoietic stem cell transplantation (HSCT),little has been studied providing a clear evidence for that. We performed EBV DNA quantitation in donors and analyzed the effect of donors’ EBV viral load on the recipients after HSCT. Methods : EBV DNA quantitation of peripheral blood in 94 healthy HSCT donors was performed by real-time PCR. We analyzed the distribution of EBV viral load in HSCT donors and EBV positivity in the recipients transplanted from donors who had detectable EBV. Results : Fifteen HSCT donors (16%) showed positive results in EBV real-time quantitative PCR. EBV viral load was below 500 copies/mL in 5 donors and above 500 (680-11,300) copies/mL in 10 donors. Five of the recipients (33.3%) transplanted from these 15 donors showed positivity in EBV PCR after HSCT. All of the EBV PCR positive recipients were transplanted from donors with viral load of >1,000 copies/mL, and 5 (71%) of 7 donors with viral load of >1,000 copies/mL was associated with posttansplant EBV PCR positivity in the recipients. Conclusions : Higher levels of EBV viral load in donors appear to be associated with EBV transmission to recipients in HSCT. EBV real-time quantitative PCR may be needed for screening EBV DNA level in HSCT donors. (Korean J Lab Med 2010;30:554-8)

발열질환 및 종양질환 소아에서 Epstein-Barr 바이러스의 감염형 분류

조현정,허재균,김종현,강진한 대한감염학회 2002 감염 Vol.34 No.3

Production of the late antigens of Epstein-Barr virus (EBV)

Hwang, Eung-Soo,Choo, Moo-Jin,Park, Jae-Won,Lim, Dong-Gyun,Park, Chung-Gyu,Kook, Yoon-Hoh,Choi, Sung-Bae,Cha, Chang-Yong 대한감염학회 1996 감염 Vol.28 No.6

목적 : EBV 감염에 대한 추적의 방법 중 환자의 혈청에서 EBV에 대한 항체를 검출하는 방법을 효율적으로 수행하기 위해 대사촉진제를 처리하여 EBV후기 항원을 많이 표출하는 방법을 이용하였다. 방법 : HR-1세포, B95-8세포와 Raji세포에 phorbol myristic acetate(PMA)와 phosphonoacetic acid(PAA)를 처리하여 EBV 후기항원을 생산 또는 억제시키고, EBV감염이 확인된 환자혈청으로 발현을 western bolt으로 검색하였다. 결과 : EBV감염이 확인된 환자혈청의 IgG항체를 이용하여 PMA에 의해 촉진되는 EBV후기 항원, 즉 43 kDa, 39 kDa 및 작은 분자량의 분획의 발현을 확인하였다. 11명의 비인강암 환자 혈청을 반응시켜 본 결과 9명의 환자혈청은 후기항원인 43 kDa와 39 kDa 분획에 IgG항체 반응을 보였다(81.8%). 43 kDa 분획에 대해 IgA 항체반응은 4명에서 양성을 나타냈다(36.4%). 작은 분자 분획에 대해서는 반응의 강약에 차이는 있으나 11명 모두에서 IgG 항체 양성 반응을 보였다(100%). IgA 항체반응은 5명에서 약하게 나타났다(45.5%). 결론 : 확인된 EBV 후기 항원에 대해 항원에 따라 차이는 있지만 비인강암환자에서 IgG 항체반응은 81.8%-100%의 반응을 나타냈고, IgA 항체반응은 36.4-45.5% 의 반응을 보여 EBV 감염과의 연관성을 시사하였다. Background : This study was performed to produce the large amount of the late antigens of EBV with the metabolic activator and to detect the antibodies to the late antigens of EBV in the sera of patients diagnosed as nasopharyngeal carcinoma. Methods : HR-1, B95-8 and Raji cells were treated with phorbol myristic acetate(PMA) and/or phosphonoacetic acid(PAA). Reactivity of the induced EBV antigens were analyzed with the sera of patients by sesten blot. Results : 43, 39 kDa and small molecular weight peptides suggested as the late antigens of EBV. IgG antibody reaction was found on 43 and 39 kDa in 9 patients and the small molecular weight peptide in 11 patients among 11 nasopharyngeal carcinoma patients. IgA antibody reaction was found on the 43 kDa peptide in 4 patients and the small molecular weight peptide in 5 patients among 11 patients. Conclusion : Although the causal relationship of nasopharyngeal carcinoma with EBV infection should be confirmed by the detection with the specific EBV genes in tumor tissue, the serological results using the induced late proteins as antigens suggest that some nasopharyngeal carcinoma in Korea be related with EBV infection.

Association between Epstein-Barr Virus In-fection and Chemoresistance to Docetaxel in Gastric Carcinoma

Hee Jong Shin,김도년,이숙경 한국분자세포생물학회 2011 Molecules and cells Vol.32 No.2

Epstein-Barr virus (EBV) is associated with human can-cers such as nasopharyngeal carcinoma, Burkitt’s lym-phoma, Hodgkin’s disease, and gastric carcinoma (GC). EBV is associated with about 10% of all GC cases globally. EBV-associated GC has distinct features from EBV-ne-gative GC. However, it is still unclear if EBV infection has any effect on GC chemoresistance. Cell proliferation assay, cell cycle analysis, and active caspase Western blot revealed that the EBV-positive GC cell line (AGS-EBV) showed chemoresistance to docetaxel compared to the EBV-negative GC cell line (AGS). Docetaxel treatment increased expression of Bax similarly in AGS and AGS-EBV cell lines. However, Bcl-2 induction was markedly higher in AGS-EBV cells, after docetaxel treatment. Although docetaxel increased the expression of p53 to a similar extent in both cell lines, induction of p21 in AGS-EBV cells was lower than in AGS cells. Furthermore, expression of survivin was higher in AGS-EBV cells than in AGS cells following docetaxel treatment as well as at basal state. EBV-lytic gene expression was induced by docetaxel treatment in AGS-EBV cells. The results suggest that EBV infection and lytic induction confers chemoresistance to GC, possibly by regulating cellular and EBV latent and lytic gene ex-pression.

Real-time PCR을 이용한 Epstein-Barr Virus DNA 검사 평가

하지혜,박용정,심정은,김현숙 대한진단검사의학회 2016 Laboratory Medicine Online Vol.6 No.1

Background: Epstein-Barr virus (EBV) is known to be the causative agent of infectious mononucleosis and EBV-related malignancies. In this study, we compared the results of three real-time PCR kits for EBV DNA assays. Methods: A total of 300 whole blood samples submitted for quantitative EBV PCR between January 2013 and September 2014 at Severance Hospital were included. The samples were tested by using the Artus EBV RG PCR Kit (Qiagen, Germany), AccuPower EBV Quantitative PCR Kit (Bioneer, Korea), and Real-Q EBV Kit (BioSewoom, Korea). Samples with discordant results between the three kits were confirmed by direct sequencing. Results: The result concordance rate and kappa coefficient (K) were 86.3% and 0.69 for Artus–AccuPower, 93.3% and 0.85 for Artus–Real-Q, and 92.3% and 0.83 for AccuPower–Real-Q, respectively. The correlations between the three kits were found to be significant, with a correlation coefficient of r=0.854 for Artus–AccuPower, -0.802 for Artus–Real-Q, and -0.977 for AccuPower–Real-Q, respectively (P<0.0001). If the real-time PCR concordant results of 258 samples and the direct sequencing results of 42 real-time PCR discordant samples were assumed to be true, the sensitivity/specificity values were 0.921/0.976 for Artus, 0.902/0.965 for AccuPower, and 0.967/1.000 for Real-Q. Conclusions: The three real-time PCR kits showed excellent sensitivities and specificities. All these kits would be acceptable for clinical and therapeutic management of EBV. However, some discordant results between the kits indicate the need for caution in clinical diagnosis and staging. Further implementation of standardized methodology would be needed for EBV DNA assays. 배경: Epstein-Barr virus (EBV)는 전염성 단구증, 악성종양의 원인으로 밝혀져 있다. 현재 국내에서 시판되는 EBV 검사 시약 중 real-time PCR을 이용한 3종류의 시약을 비교하여 보았다. 방법: 2013년 1월부터 2014년 9월까지 연세의대 세브란스병원에 EBV PCR 검사가 의뢰된 검체 중 검체량이 충분한 300검체를 임의로 선택하였다. Artus EBV RG PCR Kit (Qiagen, Germany), AccuPower EBV Quantitative PCR Kit (Bioneer, Korea), 그리고 Real-Q EBV Kit (BioSewoom, Korea)를 비교하였다. 결과: 세 가지 시약으로 검사한 결과 정성 검사 기준으로 각 검사들의 일치율과 Kappa 계수는 86.3%, 0.69 (Artus-AccuPower), 93.3%. 0.85 (Artus-Real-Q) 그리고 92.3%, 0.83 (AccuPower-Real-Q)이었다. 세 시약 결과 간의 상관성은 유의하였는데, 상관계수 r=0.854 (Artus-AccuPower), -0.802 (Artus-Real-Q), 그리고 -0.977 (AccuPower-Real-Q)이었다. 하나라도 결과가 불일치한 42검체는 sequencing 결과를 참으로 가정하여 민감도와 특이도를 구하였을 때 0.921/0.976 (Artus), 0.902/0.965 (AccuPower), 그리고 0.967/ 1.000 (Real-Q)이었다. 결론: 세 가지 real-time PCR을 이용한 EBV 검사는 모두 민감도와 특이도가 우수하였다. 임상 검사실에서는 각자의 조건에 따라 선택하면 좋을 것이다. 그렇지만 임상적 진단과 추적 검사 시 동일한 시약을 사용하는 것이 필요하며, 앞으로 표준화된 검사 방법이 필요할 것으로 생각된다.

Epstein-Barr Virus 감염 진단을 위한 특이항체검사법과 중합효소연쇄반응법의 임상적 유용성

정재림,김현숙 대한임상병리학회 2000 대한임상병리학회지 Vol.20 No.3

급성 전염성 단핵구증 환아에서 Epstein-Barr 바이러스의 감염형과 사람 백혈구 항원형 연구

한승훈,신완식,한훈,강진한,Hahn, Seung-Hoon,Shin, Wan-Shik,Han, Hoon,Kang, Jin-Han 대한소아청소년과학회 2003 Clinical and Experimental Pediatrics (CEP) Vol.46 No.5

목 적 : EBV는 전 세계적으로 다양한 질환을 일으키고, EBV는 지역과 EBV에 노출된 사람의 면역 또는 병적 상태에 따라 감염형이 달라 EBV 감염형에 관한 연구는 EBV의 역학연구와 EBV 관련 질환의 병인연구에 제일 기본적이다. EBV의 잠복감염 유전인자들에 의한 세포독성 T 림프구 면역상태는 EBV에 의한 다양한 질환의 병인적 역할이 있으며 HLA에 의하여 유발되어지는 것으로 알려져 있다. 과거에 시행한 많은 연구에서 EBV 감염과 HLA형과의 연관성은 객관적으로 입증되지 않았으나, HLA II형에 속한 항원이 EBV의 인체 내 침투에 관여한다는 사실이 최근에 보고됨으로써 이에 대한 관심이 재조명되고 있다. 이에 저자들은 EBV 감염에 의한 국내 급성 전염성 단핵구증 환아에서 주된 EBV 감염형의 분포를 확인하고, 급성 전염성 단핵구증의 발생이 HLA형과 연관성이 있는 지를 분자생물학적 분류법으로 알아보고자 이 연구를 실시하였다. 방 법 : 연구 대상은 6세에서 13세 사이의 급성 전염성 단핵구증으로 확진된 24명의 환아 이었다. 감염형의 비교 대조군은 건강한 정상 소아 중 면역혈청학적 검사로 EBV 노출이 확인된 동일한 연령의 58명이었고, HLA형 분류의 비교 대조군은 연구대상 환아와 동일한 연령의 정상 소아 200명을 대상으로 실시하였다. EBV에 의한 급성 전염성단핵구증 환아에서 중합 효소 연쇄반응법으로 EBV 감염형을 분류하여 주된 감염형을 확인하고, ARMs PCR법으로 HLA-A, B, C 항원형을, PCR-SSOP법으로 HLA-DRB 항원형을 분류하여 다음과 같은 결과를 얻었다. 결 과 : 1) EBV 감염형 결과 : 연구 대상 환아 24명에서 EBV A형이 20명(83.3%)이었고, B형은 4명(16.7%)이었다. 비교 대조군의 경우에는 58명 중 14명(24.1%)이 EBV가 검출되고 이들 중 A형은 11명(78.6%)이었고, B형이 3명(21.%)이었다. 2) HLA 형 분류 결과 : HLA I형 결과는 HLA-A24형에서 비교 대조군 200명 소아 중 69명이 양성이었고, 연구 대상 환아 24명 중 14명이 양성을 보여 RR치가 3.5724, chi-square치가 5.26, P<0.05으로 통계적 유의성을 보였다. 그러나 HLA-B, C항원형에서는 통계적으로 유의한 결과가 없었다. HLA II형 결과는 HLA-DRB1*07형에서 비교 대조군 200명 소아 중 18명이 양성이었고, 연구 대상 환아 24명 중 8명이 양성을 보여 RR치가 5.6173, chi-square치가 9.73, P<0.01로 통계적 유의성을 보였다. 결 론 : 위의 연구 결과를 통하여 국내 급성 전염성 단핵구증 환아의 EBV 감염형은 정상 비교 대조군의 소아에서 주된 감염형인 A형과 동일한 감염형임을 확인하였다. 그리고 HLA형 연구에서 HLA-A24형과 HLA-DRB1*07형이 정상 대조군과 비교하여 통계적 유의성을 보여 EBV에 의한 급성 전염성단핵구증 발생이 HLA형과 연관성이 있음을 추정하였다. Purpose : The Epstein-Barr virus(EBV), gamma herpesvirus, is an important pathogen that is widespread around the world. The EBV causes various diseases depending on the geographic location, and on the immunity or the premorbid condition of the person exposed to EBV. To evaluate EBV typing may be the most important step to figure out the pathogenesis of EBV associated diseases, and we need to re-evaluate the pathologic role of human leukocyte antigen(HLA) in developing Epstein- Barr virus associated acute infectious mononucleosis by using newly developed methods. Methods : This study included 24 children(age range : 6 to 13 years), serologically confirmed with acute infectious mononucleosis. The control group for the HLA type consisted of 200 age-matched healthy children. To classify HLA I, modified ARMs-PCR was used, while modified PCR-SSOP was utilized in typing of HLA II. Also, we performed EBV typing in study patients by using a one-step PCR. Results : The results of HLA types : In HLA class I, HLA-A24 was positive in 69 of 200 healthy children and positive in 14 of 24 patients in the study group(relative risk : 3.5724, chi-square; 5.26, P<0.05). In HLA class II, HLA-DRB1*07 was detected in 18 of 200 healthy children, and eight of 24 patients in the study group(relative risk; 506173, chi-square; 9.73, P<0.01). The results of EBV types : In the research group, 20(83.8%) of 24 patients were shedding type A virus, while 4(16.7%) were type B. Conclusion : We conclude that development of infectious mononucleosis may be associated with HLA types, and these results suggest that acute infectious mononucleosis could have hereditary traits. And we confirm that type A EBV is highly prevalent in patients with acute infectious mononucleosis in Korea. Also, our results suggest that further large scale studies, including adult groups, regarding the association between pathogenesis of EBV with HLA-DP or HLA-DQ will be warranted.

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

Dan-Min Xu,Yi-Lin Kong,Li Wang,Hua-Yuan Zhu,Jia-Zhu Wu,Yi Xia,Yue Li,Shu-Chao Qin,Lei Fan,Jian-Yong Li,Jin-Hua Liang,Wei Xu 대한암학회 2020 Cancer Research and Treatment Vol.52 No.2

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

Anti-tumor effect of Inonotus obliquus in xenograft animals with EBV+human gastric carcinoma

이슬기,조효선,Lee, Seulki,Cho, Hyosun The Microbiological Society of Korea 2016 미생물학회지 Vol.52 No.4

차가버섯(Inonotus obliquus)은 다양한 생리활성을 가진 약용버섯으로 항암, 항산화, 항염효능 등을 가진 것으로 보고되었다. EBV 양성 위암은 EBV관련 암 중 가장 빈번하게 나타나는 형태로 EBV 잠복감염이 그 원인이다. 본 연구에서는 차가버섯 주정추출물의 경구투여를 통해 EBV 양성 인간위암(SNU719) 세포주를 면역결핍 쥐에 주입 후 생기는 고형암 생성억제에 대한 효능을 연구하였다. 또한, 실험종료 후, 각각의 종양조직을 절제하여 항종양 억제기전을 탐구하였다. In vivo 종양생성억제 실험에서 차가버섯은 유의적으로 고형암 생성억제 효능을 보였다. 차가버섯이 투여된 동물유래 종양조직에서 세포자멸사와 관련된 p53, p21 및 Bax의 발현이 크게 증가하였으며, 이는 cleaved caspase-9와 cleaved Parp 발현의 상승과 동반하여 항종양 효능이 세포자멸사를 통해 나타남을 제시하였다. 또한, 이러한 항종양 효능은 세포주 내 잠복되어 있는 EBV 바이러스 유전자인 BZLF-1 및 LMP-2의 발현에도 영향을 미치는 것으로 밝혀졌다. Inonotus obliquus is a medicinal mushroom with a variety of biological activities. It has reported to have strong anti-cancer, antioxidant and anti-inflammatory properties. EBV+ gastric carcinoma is one of the most common EBV-associated cancers that were caused by latent EBV infection. In this study, we investigated the anti-cancer effects of ethanol extract of I. obliquus using in vivo xenograft animal models implanted with EBV+ human gastric carcinoma (SNU719). We also explored the molecular mechanisms responsible for its anti-cancer activity. The result indicated that the extract of I. obliquus had an anti-cancer effect in in vivo xenograft mice with EBV+ gastric carcinoma (SNU719). Extract of I. obliquus also showed a great effect on inducing the expression of p53, p21 and Bax in tumor tissue derived from EBV+ human gastric carcinoma, and these were correlated with increased expressions of the cleaved forms of caspase-9 and Parp. Also, I. obliquus attenuated the expression of viral proteins, BZLF-1 and LMP-2 in tumor tissue from EBV+ human gastric carcinoma.