Pattern of Epstein-Barr virus (EBV)-specific proteins synthesized during primary lytic infection of mouse lymphocytes by EBV

Normal mouse lymphocytes were implanted with EBV receptors and exposed to the virus of P3HR-1 strain. 5% of the cells expressed early (EA) and viral capsid (VCA) antigens as assayed by immunofluorescence 24 h after the infection. Only 0.1% of cells expressed nuclear-like antigen (EBNA) 48 h post-infection. When labelled metabolically with [35S]methionine, extracted, immunoprecipitated with EBV-positive sera, and analyzed by SDS-gel electrophoresis and autoradiography, about 20 EBV-determined proteins ranging from 19 to 165 kd were detected. Their pattern and relative quantitative expression differed from those in P3HR-1 virus superinfected Raji cells. Polypeptides of approximate molecular size 78, 72, 65, 48 and 26.5 kd were predominant in EBV-infected mouse lymphocytes. In contrast, 130, 98, 59, 50.5 and 36 kd proteins were predominant in the induced Raji cells. Our results demonstrate that rodent lymphocytes can be used for the direct biochemical analysis of EBV-translational products during primary lytic infection in normal cells.     

Generation of human monoclonal antibody-producing cell lines by Epstein-Barr virus (EBV)-transformation of B lymphocytes and somatic cell hybridization techniques

Summary The use of Epstein-Barr virus to immortalize human B cells and generate monoclonal antibody-producting B cell lines is described.     

A culture method giving substantial yields of normal nasopharyngeal epithelial cells for work with Epstein-Barr virus

A culture method, utilising a feeder layer of lethally irradiated 3T3 fibroblasts and medium supplemented with hydrocortisone, cholera toxin, and epidermal growth factor, has been elaborated for the in vitro growth of normal human nasopharyngeal epithelial cells. This method allowed the cells to be grown in vitro for periods of up to 146 days, very considerably longer than in previously reported studies, and ensured that the cultures remained largely free from contaminating human fibroblasts. It was found possible to subculture the nasopharyngeal epithelial cells through numerous passages both by dispersing monolayers into single cell suspensions and by transferring coverslip monolayers of the cells to individual Petri dishes. By combining these two methods, at least 50 replicate epithelial cultures could be produced from each tissue sample, thus providing for the first time cultured nasopharyngeal epithelial cells in quantities suitable for extensive experiments with Epstein-Barr virus.     

EB病毒对人胚鼻咽上皮细胞的转化

为观察EBV和／或促癌物四癸酸佛波醇二酯（TPA）对其的转化作用，以人胚鼻咽上皮作体外原代组织培养，采用自B95－8细胞分离的EB病毒直接感染或结合TPA处理体外培养的人胚鼻咽上皮细胞，着重观察感染细胞在半固体培养基中的集落形成率；并采用PCR扩增法探讨EB病毒是否直接进入鼻咽上皮细胞。结果显示：单独EB病毒或灭活（56℃，30分钟）EB病毒加TPA感染时，病毒不能进入细胞导致表型改变；活性EB病毒结合TPA同时处理或先用EB病毒后用TPA处理时，EB病毒能直接进入细胞并导致细胞集落形成率明显增高（P＜0．05）。从而表明EB病毒体外能部分转化人胚鼻咽上皮细胞，其转化作用依赖于TPA的存在和病毒基因组的完整。     

促癌剂协同下Epstein-Barr病毒体内外感染引起人胚鼻咽上皮的形态学改变

目的探讨在促癌剂协同下Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）对人胚鼻咽上皮的感染和转化能力。方法体外培养的人胚鼻咽上皮分别接受ＥＢＶ、ＥＢＶ＋佛波酯（ＴＰＡ）或ＴＰＡ处理，不同时间测量细胞生长晕大小，收集细胞，涂片，ＨＥ染色作形态测量；移植了人胚鼻咽组织的裸小鼠分别接受多次ＥＢＶ、ＥＢＶ＋ＴＰＡ皮下注射，处死动物后对移植物作常规病理切片观察。结果体外培养的人胚鼻咽上皮在ＥＢＶ转染后（单用ＥＢＶ或在ＴＰＡ协同下），生长能力增加，核浆比增大；在体内，ＥＢＶ感染（单用ＥＢＶ或在ＴＰＡ协同下）可引起移植于裸小鼠的人胚鼻咽组织癌变（原位癌与早期浸润癌各１例）。在体外，单独使用ＴＰＡ没有诱癌作用，但似能增加ＥＢＶ的感染率，尤其在早期；在体内，在ＴＰＡ协同下，ＥＢＶ能诱发早期浸润癌。结论提示ＥＢＶ在体外能使人胚鼻咽上皮细胞出现恶性转化倾向，及引起移植于裸小鼠的人胚鼻咽组织癌变，ＴＰＡ能促进ＥＢＶ感染和致癌作用。     

Infection of normal human thymic epithelial cells by Epstein-Barr virus (EBV) following implantation of EBV receptors

Normal human thymic epithelial cells cannot be infected and transformed by Epstein-Barr virus (EBV). Measurements using fluorescein-labelled EBV and cytofluorograph revealed that the cells do not have receptors for the virus. Following transplantation of functional EBV receptors onto epithelial cell membranes, however, the cells were infected with EBV and expressed EBV-determined nuclear antigen (EBNA). No EBV associated antigens characteristic of the lytic cycle of the virus were detected. This method of in vitro infection by transforming viruses such as EBV may provide ways of deriving functioning thymic epithelial cell lines.     

Differential expression profiling of lncRNAs related to Epstein-Barr virus infection in the epithelial cells

Epstein-Barr virus (EBV) is a prevalent γ-herpesvirus associated with a variety of cancers, including epithelial nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). Long noncoding RNAs (lncRNAs) are emerging as powerful regulators that have demonstrated to play crucial roles in cancer development. In this approach, based on genome-wide RNA sequencing, we examined the lncRNA expression profiles in four EBV genome-infected and EBV-negative 293 cells. A series of lncRNAs were found to be dysregulated in a comparative analysis. Then, eight typical lncRNAs were selected for validation of expression levels by real-time quantitative polymerase chain reaction and were detected in EBV-positive or EBV-negative GC and NPC cells. The differential expression patterns of the eight lncRNAs for validation were fundamentally identical to those revealed in RNA sequencing. Particularly, the differential expression of these lncRNAs in GC and NPC cells indicated their possible roles in EBV infection and tumorigenesis. In addition, a predicted lncRNA-microRNA-messenger RNA network suggested their potential interactions. This study reveals the first lncRNAome related to EBV infection in the epithelial cells and provides novel clues for the study of viral role in epithelial cancers through the interaction between EBV and host lncRNAs.     

Budd-Chiari syndrome and Epstein-Barr virus (EBV) associated plasmacytoma in a patient with chronic active EBV infection

A 42-year-old Japanese man with chronic active Epstein-Barr virus (EBV) infection initially responded to treatment with interleukin-2 (IL-2). Six months later he developed thrombosis in the hepatic veins, and Budd-Chiari syndrome associated with severe hepatic damage was diagnosed. He also developed a solitary EBV-positive plasmacytoma in the right femur. Since these rare complications occurred after long-term IL-2 therapy, the possibility that long-term IL-2 therapy might cause Budd-Chiari syndrome and liver damage as well as EBV-associated plasmacytoma is discussed.Abbreviations EBV Epstein-Barr virus - EBER1 EpsteinBarr virus-encoded RNA-1 - IL-2 interleukin-2 - NK natural killer - LMP latent membrane protein - LAK lymphokine activated killer     

Epstein-Barr virus (EBV)-specific cell-mediated and humoral immune responses in ataxia-telangectasia patients

As a part of studies on cell-mediated immune (CMI) responses of immunocompromised, Epstein-Barr virus (EBV)-infected patients who can or cannot restrict the proliferation of EBV-transformed B cells, we have studied 16 Turkish patients with ataxia-telangectasia (AT). Fifteen were EBV seropositive; one was seronegative. Among the seropositives, eight had no or only low anti-EBV-determined nuclear antigen (EBNA) antibody titers, while seven had normal anti-EBNA levels. EBV-seropositive and -seronegative healthy Turkish children were used as controls. We have particularly asked the question whether low EBNA antibody titers can be correlated with the level of EBV-specific and -nonspecific cell-mediated immunity. Non-EBV-specific tests included cell count, phenotypical characterization with monoclonal antibodies, assessment of natural killer (NK)-cell activity, and ability to suppress mitogen-induced immunoglobulin production. Two EBV-specific CMI tests were used: outgrowth inhibition (OI) and leukocyte migration inhibition (LMI). The majority of the patients of the low-EBNA antibody group was IgA deficient and had high levels of -fetoprotein (a-FP). Cells reacting with OKT8 monoclonal antibody predominated in both AT patient groups. In contrast, the suppressor activity was present in only a few patients and NK and interferon-activated killing (IAK) activities were normal. EBV-specific cell-mediated responses were defective in seven of eight patients in the low-anti-EBNA group and five of seven patients in the group with normal anti-EBNA titers. It is concluded that AT patients are often defective in their EBV-specific cell-mediated immune responses and with regard to their EBNA antibody levels. These defects are associated with a predominance of T cells reacting with OKT8 monoclonal antibody.     

EB病毒和ConA诱导抑制性T细胞对EB病毒感染B细胞作用探讨

本文探讨了用灭活的EB病毒(EBV)和ConA诱导产生的抑制性T细胞(Ts),对EBV感染自身B细胞的影响,结果表明,EBV抗原诱导产生的抑制性T细胞(Ts)能使EBV感染B细胞中的EBNA阳性细胞数,~3H-TdR掺入量和IgA、IgG及IgM分泌量减少;而ConA诱导产生的Ts则使EBNA阳性细胞数和~3H-TdR掺入量增加,但三种Ig含量无明显变化(P>0.05)。结果提示前者对EBV感染B细胞的激活,增殖和分化均有明显抑制作用,而后者的作用则相反,具有明显促进EBV感染B细胞的作用。     

Epstein-Barr virus (EBV)-associated undifferentiated carcinoma of the parotid gland

A case of undifferentiated carcinoma of the salivary gland occurring in the parotid gland of a southern Chinese was reported. Tumour cells showed immunofluorescence for Epstein-Barr virus (EBV)-associated nuclear antigen, and DNA hybridization demonstrated the presence of EBV-DNA in tumour tissue. The findings in this case, together with previous reports, suggest a causal relationship between EBV and salivary gland carcinoma. The relationships between EBV and undifferentiated epithelial tumours of the salivary glands, nasopharynx and thymus are also discussed.     

Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin''s disease is frequently associated with CR2 (EBV receptor) expression

We studied 44 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non-radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed-Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV-receptor CR2 (CD 21) on Reed-Sternberg cells was studied using the same techniques and two different monoclonal anti-CD 21 antibodies. CR2 could be detected on a substantial number of the Reed-Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti-CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed-Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.     

CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection

The Epstein-Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV-infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme-based and the other Fas (CD95)/Fas ligand (FasL)-based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8⁺ T-cells increased in number, but CD56⁺ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell-restricted intracellular antigens (TIA-1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA-1 positive cells was smaller than that in CAEBV. Fas-positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL-positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin- and/or granzyme-positive cells was small in number in both CAEBV and IM cases. In double-staining FasL and EBV in situ hybridization, FasL-positive EBV-infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV-infected lymphocytes may evade immune attack through the expression of FasL.     

Infection of the human T-cell-derived leukemia line Molt-4 by Epstein-Barr virus (EBV): induction of EBV-determined antigens and virus reproduction

We have reported previously that human T-cell-derived Molt-4 cells become susceptible to Epstein-Barr virus (EBV) infection after implantation of functional EBV receptors into the plasma membranes of Molt-4 cells (Volsky et al., 1980). In the present work, we expand this finding by analyzing the following: (i) Virus adsorption vs viral penetration—using [³H]thymidine-labeled EBV, we demonstrate that the virus could adsorb to both the untreated and the receptor-implanted Molt-4 cells. However, only the altered cells became susceptible to EBV penetration followed by the viral antigen synthesis; (ii) EBV substrain specificity of infection—EBV P3HR-1 virus induced the nuclear (EBNA), early (EA), and virus capsid (VCA) antigens, whereas EBV B-95-8 virus induced only EBNA; (iii) virus reproduction—in situ hybridization was used to demonstrate the EBV-DNA synthesis in the P3HR-1 virus-infected cells. In addition, immature herpes-like particles were observed in electron micrographs of infected cells. It is concluded that EBV infection of human T-cell-derived Molt-4 cells may lead to a full viral-lytic cycle in a portion of infected cells. The results suggest, however, that the primary EBV productive infection may not necessarily involve any immunofluorescence-detectable EBNA synthesis.     

EBV utilizes a unique activation pathway for the transformation of human B cells

EBV growth-transforms primate B lymphocytes and directly causes mono/multiclonal B cell lymphomas in vulnerable hosts. In this report we demonstrate that the degree of B cell transformability is not quantitatively determined at the level of either the saturable, transformation-prerequisite virus receptors or of the actual viral cell entry process. Instead, post-receptor binding events [Na+/H+ exchange, Ca2+ flux, tyrosine phosphorylation of two proteins (55-60/130-140 kd)] were identified as critical determinants of transformability. The presence of competent virus in transformable cells was per se insufficient for transformation: blockade of Ca2+ fluxes (or the antiport) generates virus-loaded cells that express viral genes but remain untransformed. Delayed induction by ionomycin of appropriately sized Ca2+ fluxes ([Ca2+]i greater than 180 less than 400 nM) re-starts transformation processes in EGTA-blocked, virus-loaded cells, perhaps providing a model for the study of virus re-activation. Overall, EBV induces unique cellular activation events different from non-oncogenic lymphocyte mitogens/activators, and, given the oncogenic potential of transformed cells in susceptible hosts, we hypothesize that these events describe a novel oncogenic transformation pathway.     

Immunization of human lymphocytes with Epstein-Barr virus capsid antigens in vitro

Research Center for Development and Introduction of Modern Methods of Molecular Diagnosis, Ministry of Health of the USSR, Moscow. (Presented by Academician of the academy of Medical Sciences of the USSR A. I. Borob'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 1, pp. 62–64, January, 1991.     

The Epstein-Barr virus BMRF-2 protein facilitates virus attachment to oral epithelial cells

We previously reported that BMRF-2, an Epstein-Barr virus (EBV) glycoprotein, binds to beta1 family integrins and is important for EBV infection of polarized oral epithelial cells. To further study the functions of BMRF-2, we constructed a recombinant EBV that lacks BMRF-2 expression by homologous recombination in B95-8 cells. We found that lack of BMRF-2 resulted in about 50% reduction of EBV attachment to oral epithelial cells, but not to B lymphocytes, suggesting that BMRF-2 is critical for EBV infection in oral epithelial cells, but not in B lymphocytes. In polarized oral epithelial cells, infection rate of the recombinant EBV virus was about 4- to 8-fold lower than the wild-type B95-8 virus. Cell adhesion assays using the BMRF-2 RGD peptide and its RGE and AAA mutants showed that the RGD motif is critical for BMRF-2 binding to integrins. These data are consistent with our previous observation that interactions between EBV BMRF-2 and integrins are critical for infection of oral epithelial cells with EBV.