Epstein-Barr virus-associated complement-fixing and nuclear antigens in Burkitt lymphoma biopsies

Cells from Burkitt lymphoma (BL) biopsies were examined for Epstein-Barr virus (EBV)-associated antigens by complement fixation (CF) tests and by the anti-complement immunofluorescence (ACIF) test. In CF tests, anticomplementary factors made it difficult to test all the biopsies available. However, biopsies from 19 patients were effectively tested and 12 of these (including two from one patient) contained antigen reacting with a battery of human sera with antibody to EBV but not with sera lacking such antibody. Technical difficulties prevented further characterization of the EBV-related antigens in the biopsies. Application of the ACIF test to BL revealed the presence of EBV-related nuclear antigen in biopsies from 11 of 13 patients. Absorption studies indicated that the nuclear antigens of the biopsies were closely related antigenically to the EBV-determined nuclear antigen (EBNA) previously described in lymphoblastoid cell lines. It is concluded that cells of BL biopsies may contain EBNA in addition to the EBV-related membrane antigen previously described. The results provide further evidence that BL cells from African patients resemble non-producer lymphoblastoid cell lines in containing EBNA and therefore appear to be transformed by EBV.     

Discordant Epstein-Barr virus nuclear antigen (EBNA) antibody patterns in nasopharyngeal carcinoma

Epstein-Barr virus nuclear antigen (EBNA) preparations from three sources were tested with sera from normal individuals and patients with Hodgkin's disease, breast carcinoma, Burkitt's lymphoma, and American and Chinese nasopharyngeal carcinoma. Individual sera with discordant antibody patterns were noted in all groups. Sera from both NPC groups gave significantly higher anti-EBNA titers on cell lines converted with P3HR-1 or B95-8 virus compared with anti-EBNA titers on Raji cells. Anti-EBNA titers of Chinese NPC sera showed no correlation among the three EBNA sources, while all other groups had highly correlated titers. Cross-absorption experiments present evidence for more than one antigenic determinant on EBNA. These results suggest an additional parameter for distinguishing Chinese NPC from other EBV-related disorders.     

Detection of a nuclear, EBNA-type antigen in apparently EBNA-negative Herpesvirus papio (HVP)-transformed lymphoid lines by the acid-fixed nuclear binding technique.

In agreement with the findings of previous authors, we could not detect a virally determined nuclear antigen in Herpesvirus papio (HVP)-transformed baboon lymphoid lines by anticomplementary staining in situ, as for EBNA. However, by means of our recently developed acid-fixed nuclear binding technique an EBNA-like antigen could be readily demonstrated, after extraction from both producer and non-producer lines. We propose to designate the antigen as HUPNA. It can be detected by a human anti-EBNA antibody, suggesting cross-reactivity, if not identity, between EBNA and HUPNA. HVP-DNA carrying non-producer lines, negative for in situ ACIF stainability but capable of yielding HUPNA by the nuclear binding technique, can be superinfected with EBV, with brilliant EBNA expression as the result, suggesting that the defective in situ staining is a property associated with the baboon HVP, rather than the baboon lymphoid cell per se.     

Antibody response against the Epstein-Barr virus-coded nuclear antigen2 (EBNA2) in different groups of individuals

Specific antibody responses against the 2 major subcomponents of EBNA, EBNA1 and EBNA2 were evaluated, in order to study whether this serological study was beneficial compared to classical EBV serology. During this investigation, 491 sera, obtained from blood donors and patients with Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), infectious mononucleosis (IM), Hodgkin's disease, renal transplantation, rheumatoid arthritis and Human Immunodeficiency Virus (HIV) infection, were tested. While the anti-EBNA1 response followed the classical anti-EBNA/Raji response (99% of anti-EBNA/Raji-positive sera also recognize EBNA1), the anti-EBNA2 response was much less frequent and did not correlate with either anti-EBNA/Raji or anti-EA antibodies. In a control population, 8% of individuals had antiEBNA2 antibodies at titers greater than or equal to 10. The percentage was 45% in NPC and 38% in EBV-associated BL; thus, although not detected in all patients with EBV-associated tumors, anti-EBNA2 serology might be a useful marker in BL and NPC. No antibody was detected in the early course of IM, but in rheumatoid arthritis and in HIV-infected patients, the percentage of positive individuals reached 54 and 68, respectively. Seroconversion to EBNA2 was noted in a few cases, including renal transplant recipients, AIDS patients, and complicated IM. This suggests that in these situations, EBNA 2 serology might represent a useful marker related to modulation of the immune status or EBV reactivation.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

Elevated expression of ICAM1 (CD54) and minimal expression of LFA3 (CD58) in Epstein-Barr-virus-positive nasopharyngeal carcinoma cells.

Undifferentiated nasopharyngeal carcinoma (NPC) is a remarkable entity among human tumors because of its constant association with the Epstein-Barr virus (EBV). Malignant epithelial cells harbor the EBV genome and often express at least 2 species of latent EBV protein (EBNA1 and LMP1). Despite the massive presence of tumor-infiltrating lymphocytes, NPC cells obviously escape immune surveillance directed to EBV antigens. Previous investigations carried out on EBV-positive Burkitt lymphoma (BL) cells have shown that this fact may be partially accounted for by a lack of expression of ICAM1 (CD54) and LFA3 (CD58). ICAM1 and LFA3 have therefore been investigated in fresh NPC biopsies and transplanted NPCs. With only 1 exception out of 9 cases, NPC cells appear to express high levels of ICAM1 and low levels of LFA3. This is a complete inversion of the pattern observed in normal epithelial cells in vivo. Additional investigations will be required to determine to what extent these characteristics affect T-cell interactions with NPC cells, specially in the process of EBV-antigen recognition.     

Study of Epstein-Barr virus-determined nuclear antigen (EBNA) by chromatography on fixed cell nuclei.

Low ionic strength (50 to 100 mM NaCl) and pH 6.0 were found to be optimal conditions for in vitro conversion of Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA)-negative nuclei to EBNA-positive nuclei by addition of the complement-fixing (CF) antigen extracted from Raji cells. In vitro conversion of nuclei to EBNA-positively was sensitive to DNase but not to RNase treatment. This suggests that nuclear DNA is a specific target substance to which EBV-CF antigen binds. If nuclei were fixed with methanol/acetic acid and subsequently treated with 0.6 M NaCl, EBNA could be eluted from in vitro-converted Ramos nuclei with 0.3 and 0.4 M NaCl. The same conditions were also found to be optimal for the adsorption and elution of EBV-CF antigen in DNA-cellulose chromatography. This indicates that the DNA-binding properties of EBNA antigen can be studied by "chromatography" on fixed nuclei followed by the ACIF test. The obvious advantages of this method over chromatography on DNA-cellulose are its simplicity, the possibility of testing many samples in one experiment and, especially, the use of minimal amounts of material. Significant differences in elution patterns for EBNA were found when nuclei derived from different cell lines (Ramos, Raji, and P3HR-1) were converted in vitro to EBNA-positivity. EBNA is eluted from in vitro-converted nuclei of EBV genome-positive P3HR-1 cells at an almost 0.1 M higher concentration of NaCl than is necesssary for a similar degree of elution from nuclei of EBV genome-negative Ramos cells.     

Association of Epstein-Barr virus with nasopharyngeal carcinoma in Alaskan native patients: serum antibodies and tissue EBNA and DNA

Biopsy specimens from Alaskan Native patients with nasopharyngeal carcinoma (NPC) and from other patients seen on the otolaryngology service were tested for Epstein-Barr virus-specific DNA and nuclear antigen (EBNA). Serum samples from both groups were tested for various EBV-related antibodies. EBV DNA and EBNA results were in agreement in 29 of 31 tissue specimens tested by the two methods. Ten of 11 biopsies containing NPC cells were positive for EBV DNA. Two NPC patients had biopsies that showed only atypical epithelium but were also positive for EBV DNA or EBNA. The other tissue specimens were negative except for biopsies from two patients: one with a parotid gland lymphoepithelial lesion; another with undifferentiated carcinoma of salivary gland origin.     

CpG methylation of viral DNA in EBV-associated tumours

In EBV-immortalized lymphoblastoid cell lines (LCLs) a small number of "latent" proteins are expressed. These are the EBV nuclear antigens, EBNAs 1-6, and a latent membrane protein, LMP. We have investigated the expression of these proteins in a variety of EBV-associated tumours and cell lines. Whereas transplant and B-cell lymphomas from cotton-top tamarins appear to express the full range of antigens found in LCLs, we and others have found that in Burkitt's lymphomas (BL) and a nasopharyngeal carcinoma (NPC) isolate, EBNA expression is restricted to EBNA-I. (In NPC, but not in BL, LMP may also be expressed). In order to ask what restricts the expression of EBNA 2-6 in NPC and BL cells it seemed reasonable to consider the possibility that the DNA sequences normally regulating expression of these antigens could be chemically modified. In this analysis, a tight inverse correlation between methylation of CpG dinucleotides in the 5' flanking region of the EBNA-2 gene and the expression of EBNAs 2-6 has been revealed. In the NPC tumour, CpG methylation within the gene is also observed, as is specific methylation over the EBNA-I region I and II binding sites (in oriP). The significance of these observations is considered.     

Relationship between Epstein-Barr virus (EBV) DNA and the EBV-determined nuclear antigen (EBNA) in Burkitt lymphoma biopsies and other lymphoproliferative malignancies

Twenty-six of 27 African Burkitt lymphomas with histologically confirmed diagnosis contained relatively large amounts of EBV DNA (10–101 viral genomes per cell), as determined by nucleic acid hybridization. Twenty-five of the 26 EBV DNA-positive lymphomas contained the EBV-determined nuclear antigen, EBNA, in the majority of the nuclei. Technical reasons may have accounted for the apparent EBNA-negativity of one EBV DNA-positive biopsy. Four African lymphoma biopsies, one with a definite diagnosis of Burkitt's lymphoma and three with a questionable diagnosis of the same disease, were all EBV DNA- and EBNA-negative. The same was true for a collection of Swedish cases of Hodgkin's disease, lymphocytic lymphoma, chronic lymphatic leukemia and some other lymphoproliferative malignancies. Thus, there is excellent agreement between the presence of EBV DNA and of EBNA in tumor biopsies. The EBNA antigen test therefore appears a relatively simple way of testing for the presence of the virus genome, provided it is carried out with appropriate controls. Several of the EBV-genome and EBNA-negative cases came from patients with high serum titers of EBV antibodies. It is concluded that the virus does not really travel along with malignant lymphomas as a passenger in the seropositive patients. In comparison with other lymphomas, African Burkitt's lymphoma of the high endemic areas is unique in that the tumors (with rare exceptions) represent the proliferation of an EBV-genome carrying clone. These findings stress the necessity to distinguish between EBV-seropositive status and evidence for EBV-genome-carrying neoplastic cells.     

Demonstration of Epstein-Barr virus-associated nuclear antigen in nasopharyngeal carcinoma cells from fresh biopsies

Fresh nasopharyngeal carcinoma (NPC) biopsies were treated in several ways to yield satisfactory cell preparations for detection of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) by anti-complement immunofluorescence tests. If the cells were well dispersed (trypsinization or extensive mechanical dispersal) few or none of them were EBNA-positive. In contrast, if the biopsy preparations contained small to moderately sized tissue fragments (touch preparations or limited mechanical dispersal), nuclear staining was detected in nearly every cell at the margin of the fragments or in cell sheets protruding from them. The stained cells corresponded to the carcinoma cells when compared to histologically stained replicate cell preparations. Nuclear staining was obtaining with anti-EBNA positive sera [healthy donors, NPC patients, convalescents from infectious mononucleosis (IM)], shown to be free of antibodies to other nuclear antigens, but not with anti-EBNA negative sera (healthy donors or patients in the early acute phase of IM). These results confirm and extend previous reports that the carcinoma cells harbor EBV genomes. The implications of these findings are discussed.     

V-val突变型EB病毒核抗原1的功能研究

背景与目的:EB病毒核抗原1(Epstein-Barr viral nuclear antigen 1,EBNA 1)对于维持EB病毒的潜伏感染有重要作用。V-val变异型的EBNA1与鼻咽癌有密切的相关性。本研究旨在比较原型和V-val变异型的EBNA1基因在上皮细胞中的功能的差异。方法:用PCR方法扩增出原型和V-val变异型EBNA1基因的全长并克隆到pGFP载体上,转染HEK293细胞,检测两种亚型的EBNA1蛋白的表达对细胞生物学性状的影响。以含有EB病毒增强子FR序列的荧光素酶载体作为报告基因,比较两种亚型的EBNA1基因对质粒的转录激活能力。结果:原型和V-val变异型EBNA1基因的表达对HEK293细胞的生长速度没有明显影响,但表达原型EBNA1的细胞的克隆形成能力明显低于V-val亚型。在裸鼠致瘤实验中,接种表达原型和V-val变异型EBNA1细胞的实验组均未见肿瘤形成。但是在瞬时转染实验中,表达V-val变异型EBNA1基因的HEK293细胞的荧光素酶活性明显高于原型EBNA1基因。结论:原型和变异型EBNA1均未发现有明显的转化细胞的能力,但是V-val变异型EBNA1蛋白与原型相比,其转录激活的功能明显增强。     

Functional advantage of NPC-related V-val subtype of Epstein-Barr virus nuclear antigen 1 compared with prototype in epithelial cell line

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC) and the viral nuclear antigen 1 (EBNA1) plays a crucial role in viral latency. Three EBNA1 subtypes, P-ala, V-thr and V-val have been detected from healthy carriers in Guangzhou area. A close relation of V-val EBNA1 with NPC was suggested by its preference to infect NPC cells. We therefore investigated the functional difference among these three EBNA1 subtypes in human epithelial cell line. The three coding sequences of the EBNA1 subtypes were cloned into the pGFP-C2 vector, and transfected into 293 cells, respectively. Effect of EBNA1 expression on cell proliferation was examined. The maintenance activity and expression level of EBNA1-plasmid in 293 cells were evaluated by using GFP as a reporter. The expression of P-ala, V-thr or V-val EBNA1 had no effect on 293 cell growth, while the relative average intensity of fluorescence after 14-day selection in V-val-EBNA1/293 cells was statistically higher than P-ala-EBNA1/293 (P<0.05, t test). We suggest that V-val EBNA1 with the functional advantage compared with prototype shown in this study might contribute to the tumorigenesis of NPC by increasing the expression of itself or other viral or cellular genes.     

Serum and salivary IgA antibodies against a defined epitope of the Epstein-Barr virus nuclear antigen (EBNA) are elevated in nasopharyngeal carcinoma.

The Epstein-Barr virus nuclear antigen I (EBNA I) is the only latent EBV antigen consistently expressed in malignant tissues of the nasopharynx. A 20-amino-acid synthetic peptide, p107 contains a major epitope of EBNA I. We tested sera from 210 patients with nasopharyngeal carcinoma (NPC) and from 128 normal individuals (NHS) for IgA antibodies to p107 using an enzyme-linked immunosorbent assay (ELISA). Whereas 191/210 (91%) of NPC patients had IgA antibodies to p107, only 17/128 (13.3%) of NHS had such antibodies and only 6/57 (10.5%) of sera from patients with malignancies other than NPC had IgA-p107 reactivity. Thirty-nine salivary samples from 46 NPC patients (84.8%) also contained IgA-p107 antibodies whereas only 3/42 (7.1%) of normal saliva samples were IgA-p107 positive. The results suggest that IgA antibodies to EBNA I may become a useful, easily measurable, marker for NPC.     

Evaluation of epstein-barr virus-associated nuclear antigen with various human cell lines

Critical evaluation of the anti-complement immunofluorescence (ACIF) test for Epstein-Barr virus-associated nuclear antigen (EBNA) in known EBV-associated cell lines was carried out. The ACIF procedure, in which the cell smears were thoroughly air-dried and fixed with carbon tetrachloride (for 15 min at room temperature), provided reproducible staining results with minimum variations. The fixed smears could be stored for at least 5 weeks at ?8° C without loss of reactivity. On the basis of the above evaluation, a total of 59 human cell lines, which had unknown or uncertain association with EBV, were examined for the presence or absence of EBNA by the ACIF procedure. All of the cell lines tested could be judged positive or negative for EBNA. All of 21 lines growing as monolayer or in a mixed state of monolayer and suspension, were negative. Of 38 lines grown in suspension, 36 lymphoblastoid lines were positive but the remaining 2 lines (seemingly not lymphoblastoid) were negative.     

Antiviral agent cidofovir decreases Epstein-Barr virus (EBV) oncoproteins and enhances the radiosensitivity in EBV-related malignancies

The Epstein-Barr virus (EBV) is involved in the carcinogenesis of several human cancers such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Given the consistent role of EBV in transformation and maintenance of malignant phenotype, antiviral strategies provide an attractive approach to target EBV-expressing cells. In that aim, we have tested the Cidofovir, which is an acyclic nucleoside phosphonate analog known to exert an antiproliferative activity in some human virus-related tumors. Here, we show that Cidofovir induces a downregulation of the EBV oncoprotein LMP1 associated with a decrease of the antiapoptotic Bcl-2 and an increase of the proapoptotic Bax protein in Raji (BL) and C15 (NPC) cells. Using BL cell line BL2 B95-8 (BL2 infected with the B95.8 strain of EBV), we addressed the relation between EBV genome expression and modulation of viral oncoproteins by Cidofovir and/or ionizing radiation (IR). Cidofovir was able to significantly reduce LMP1 and EBNA2 mRNA and protein expression. This effect was associated with inhibition of proliferation, stimulation of apoptosis, and decrease of Bcl-2 expression in BL2 B95.8 cells. In addition, Cidofovir enhanced the radiation-induced apoptosis and the radiosensitivity through the proteolytic cleavage of death effectors caspase-9 and -3, which was specifically induced by combined treatment in EBV-positive cells compared to their negative counterparts. Furthermore, the combined treatment in nude mice led to a complete tumor remission without increasing toxicity in two human EBV-related cancer xenografts (Raji and C15). These results provide the basis for a novel anticancer strategy to enhance the therapeutic ratio of IR in EBV-related cancers.     

Burkitt-like lymphoma in an English child: characterisation of tumour biopsy cells and of the derived tumour cell line

An eight year old English boy presented with an abdominal undifferentiated 'Burkitt-like' lymphoma. Lymphoma cells from ascitic fluid were cultured on a human embryo fibroblast feeder layer and, after a short lag period, a cell line (DH-BL) was established which, like the original tumour, was both negative for the Epstein-Barr nuclear antigen (EBNA) and expressed a monoclonal pattern of surface immunoglobulin (alpha lambda). DH-BL also possessed the Burkitt-related 8:14 chromosome translocation in all metaphases analysed; no other chromosomal abnormalities were present. The cell surface phenotype of the original biopsy cells and the cultured tumour cells in early passage were investigated using a panel of monoclonal antibodies to B lineage-associated antigens. These antibodies had recently been used to characterise African 'endemic' Burkitt's lymphoma (BL) biopsy cells and their derived cell lines. The cell surface phenotype of this English EBNA negative Burkitt-like lymphoma biopsy was indistinguishable from that previously shown by biopsies of EBNA positive endemic BLs. It therefore appears that both the endemic and sporadic forms of BL, as illustrated by this case, may be derived from the same subset of progenitor cells.     

The Epstein-Barr virus determined nuclear antigen is composed of at least three different anitgens

The EBV-determined nuclear antigen, EBNA, is the only known viral product to be regularly detected in all EBV-transformed cells. The anticomplement immunofluorescence (ACIF) staining detects an EBV-specific nuclear reaction that has recently been shown to be due to at least 2 different proteins, EBNA-1 and EBNA-2, encoded by different parts of the viral genome. We now report the existence of a third antigen of the EBNA complex, designated as EBNA-3. Serum from a patient with chronic infectious mononucleosis contained no detectable antibodies to EBNA-1 and had only a low EBNA-2 antibody level. Nevertheless, it gave an EBV-specific nuclear reaction of normal intensity and stained EBNA-2-positive and EBNA-2-negative EBV-carrying lines equally well. Immunoblotting with the same serum identified a new EBV-specific nuclear protein of 143-157 kDa.     

Persistence of Epstein-Barr viral nuclear antigen (EBNA) in cells entering the EB viral cycle.

It was shown by double immunofluorescence studies that Epstein-Barr viral nuclear antigen (EBNA) was preserved in EBV-infected cells after they had entered the productive viral cycle, as signalled by the appearance of the early antigen (EA)complex. A nuclear component of the EA comples could be clearly distinguished from EBNA with regard to antigenic specificity.