EBV-positive Cell Lines Research Articles

Immunovirological studies of fatal infectious mononucleosis in a patient with X-linked lymphoproliferative syndrome treated with intravenous immunoglobulin and interferon-α

We have studied a 19-year-old male with X-linked lymphoproliferative syndrome (XLP) and infectious mononucleosis (IM) who was treated with high-dose immunoglobulin (500 mg/kg/day) and recombinant interferon (IFN)-α (2 × 10 6 IU/m 2/day). Fulminant hepatitis was delayed; however, virus-associated hemophagocytic syndrome, cholestatic jaundice, and renal failure occurred terminally. Initially, nonspecific natural killer (NK) cell activity against K562 cells was normal but it gradually decreased. Although reactive T cells were markedly increased in his blood during the acute phase, spontaneous EBV-positive cell lines were easily established. Additionally, his mononuclear cells produced IFN-γ but not IFN-α prior to treatment. Based on results of in vitro studies, we conclude that both IFN-α and IFN-γ production are likely necessary for inhibiting EBV immortalization in vitro. Both IFN-α and -γ were produced in cultures of B95-8 EBV-infected mononuclear cells from EBV-seropositive healthy individuals. These results suggest that defective EBV-specific cytotoxic T cell activity accompanied with defective or discordant IFN-α and -γ production permitted the development of fatal IM in this patient. Combined treatment with immunoglobulin and IFN-α appeared to be partially effective during the early stage of this disease.

Susceptibility of EBV-carrying B cell lines to infection by HIV-1: variability of production of progeny virus and expression of viral antigens

We have examined 3 different EBV-carrying B cell lines, in terms of ability to be super-infected by the human immunodeficiency virus (HIV-1), and have followed these lines, after infection by HIV-1, over a period of 3 months. We found significant variation among different HIV-1 strains in terms of the multiplicity of infection required to initiate infection in these EBV-positive cell lines. Persistent infection by HIV-1 in the absence of detectable cytopathic effects could be demonstrated, as evaluated by a variety of techniques, including reverse transcriptase assay and immunofluorescence. The results indicate also that all of these cell lines produced progeny HIV-1 intermittently, with large amounts of virus production on some days but not others. In contrast, they were all able to continuously express p24.

Epstein-Barr virus latent membrane protein: induction of B-cell activation antigens and membrane patch formation does not require vimentin.

The latent membrane protein (LMP) of Epstein-Barr virus (EBV) forms patches associated with the vimentin intermediate filament system in EBV-transformed lymphoblastoid cell lines, EBV-infected Burkitt's lymphoma cells, and LMP-transfected, EBV-negative Burkitt's lymphoma cells. By gene transfer, LMP induces the expression of vimentin and B-cell activation antigens in EBV-negative Burkitt's lymphoma cells. We have now expressed LMP in an EBV-positive Burkitt's lymphoma cell line, Daudi, which does not express any LMP or vimentin. In these Daudi transfectants, LMP still formed plasma membrane patches in the absence of vimentin. LMP did not resist nonionic detergent extraction in Daudi cells as it does in vimentin-expressing cells. LMP still retained functional activity as judged by induction of B-cell activation antigens. These data indicate that LMP can form plasma membrane patches and induce B-lymphocyte activation independent of vimentin association.

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.

Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7 8 (87%) EBV-negative lymphoma cell lines of American origin and in 6 6 (100%) normal lymphoblastoid cell lines, but in only 1 6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5 5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.

The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent versus productive EBV promoters

The Epstein-Barr virus (EBV) BZLF1 gene product is thought to mediate the disruption of latent EBV infection. We have examined the regulatory effects of BZLF1 by studying its transactivating effects on seven different EBV promoters. We find that whereas the BZLF1 gene product increases the activity of the two early promoters, BMLF1 and BMRF1, it decreases the activity of three latent promoters (the BamHI-C and BamHI-W Epstein-Barr nuclear antigen promoters and the latent membrane protein promoter). The BZLF1-induced changes in promoter-directed chloramphenicol acetyltransferase activity occur in EBV-negative as well as EBV-positive cell lines and are accompanied by a similar change in chloramphenicol acetyltransferase mRNA. Deletion analysis of the BamHI Z fragment indicates that in a portion of the amino-terminal half of the BZLF1 gene product (amino acids 24 to 86) is not essential for positive transactivating effects but is required for down-regulating effects. Thus, different domains of the same EBV immediate-early gene product can either increase the function of EBV promoters active in productive infection or decrease the function of key promoters active in latent infection.

Two families of sequences in the small RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B

DNA sequence analysis was carried out on the 1-kilobase SacI-EcoRI region of the EcoRI J fragment of four strains of Epstein-Barr virus (EBV) (MABA, P3HR-1, FF41, and NPC-5), and the sequences were compared with the prototype sequence from strain B95-8. Ten single-base changes which grouped the strains into two families (1 and 2) were found. Restriction endonuclease polymorphisms predicted from the sequences were used to classify the EBV DNA from a further 26 EBV-positive cell lines into these two families. The EBNA-2 types (A or B) of the strains were found to correlate with the J region type; EBNA-2 type A DNA regularly contained J region sequence type 1, while EBNA-2 type B DNA generally carried J region sequence type 2. These data are consistent with the notion of there being two distinct families of EBV with discrete, conserved differences in DNA sequence.

Epstein-Barr virus-specific transcription in normal and malignant nasopharyngeal biopsies and in lymphocytes from healthy donors and infectious mononucleosis patients.

Cytoplasmic RNA was prepared from biopsy material obtained from the nasopharynx of normal individuals and nasopharyngeal carcinoma (NPC) patients. Similar RNA preparations were prepared from lymphocytes of healthy donors and infectious mononucleosis patients. RNA was radioactively labelled in vitro and hybridized to cloned fragments of B95-8 Epstein-Barr virus (EBV). In all seropositive cases the minimum pattern of EBV-specific RNA expression was like that observed previously in latently infected, EBV-positive lymphoblastoid cell lines or Burkitt's lymphoma biopsies. A novel observation was that all of the control biopsies from normal nasopharynxes expressed EBV-specific RNA, some of which appeared to be associated with a more active state of EBV infection. The pattern of expression in NPC patients was similar to that of the normal donors but showed some variations in complexity. In general the virus-specified small RNAs were not present in normal nasopharyngeal tissue but were expressed in the NPC biopsies.

Epstein-Barr virus mediates a switch in responsiveness to transforming growth factor, type beta, in cells of the B cell lineage.

The functional performance of the transforming growth factor beta (TGF beta), appears to depend on the target cell phenotype as well as in vitro culture conditions. We show here that Epstein-Barr virus (EBV)-infection may induce a change in responsiveness to TGF beta, since TGF beta inhibits traverse of the cell cycle of activated normal human B cells, but promotes cell proliferation of EBV-positive Burkitt's lymphoma cell lines as well as EBV-infected B cells. We present evidence that the switch in the responsiveness to TGF beta is mediated by EBV infection, irrespective of the proliferative status of target cells, and thus may contribute to the initiation as well as the maintenance of certain B cell neoplasias.

Induction of Oligo-2',5'-adenylate Synthetase in Human Lymphoid Cells Treated with 5-Azacytosine and 5-Iododeoxyuridine

Oligo-2',5'-adenylate synthetase activity was investigated in several human lymphoblastoid cell lines of B cell origin treated with reagents having effects on gene expression, 5-azacytosine (5AZct), 5-azacytidine (5AZcd) and 5-iodo-2'-deoxyuridine (IUdR). Enzyme activity did not increase in cells treated with 5AZcd, but the other reagents induced significant levels of activity. Interferon (IFN) activity was not detected in culture fluids of cells treated with 5AZcd or 5AZct. On the other hand, treatment of cells with IUdR led to the production of 5 to 10 units/ml IFN by NC-37 and Raji cells, but not by other human B cell lines. Enzyme induction by IUdR occurred in IFN-producing cells, NC-37 and Raji, exposed to anti-IFN-alpha sera and also in IFN-non-producing human B cells. The effect of 5AZct on enzyme induction was observed only after cultivation of cells for more than 1 month. This compound could not induce enzyme activity in Epstein-Barr virus (EBV)-negative cells, BJAB, but only in the EBV-positive cell lines NC-37 and Raji. In contrast, treatment of cells with IUdR for only 3 days induced the enzyme in all human lymphoblastoid cell lines of B cell origin except P3HR-1, but not in other human cell lines, FL, HeLa and K562. Oligo-2',5'-adenylate synthetase activity in P3HR-1 cells, which produce EBV particles, was hardly affected by IUdR or IFN treatment.

Effect of nickel sulfate on cellular proliferation and Epstein-Barr virus antigen expression in lymphoblastoid cell lines

Nickel is found in high levels in the environment of the high-risk areas for nasopharyngeal carcinoma (NPC) of China. Epstein-Barr virus (EBV) is associated with NPC and the interaction of nickel and EBV may be a contributive cofactor to the development of NPC. The study of the in vitro effect of nickel sulfate on cell proliferation and EBV-antigen expression demonstrated that nickel increases cell proliferation of some EBV-positive lymphoblastoid cell lines and increases early antigen expression of Raji cells. Nickel exerted variable effects on viral capsid antigen (VCA): increasing VCA-positive cells in B95-8 cells while decreasing VCA in P3HR-1 cells. It is proposed that the uptake of nickel in NPC high-risk areas could be one of the factors responsible for cancer development in the nasopharynx in China.

Characterization of an Epstein-Barr virus-induced thymidine kinase.

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.

新EBV(Epstein-Barr virus)陽性上皮系雑種腫瘍細胞の細胞増殖およびEBV関連抗原の検討とそれらへのインターフェロン効果

An EBV-positive epithelial hybrid tumor cell line (KB /P3 ) was newly established by cell fusion of EBV-negative 8-azahypoxanthine-resistant epithelial tumor cells (KB-AH) with EBV-positive lymphoblastoid cells (P3HR-1). With this new hybrid tumor cell line (KB/P3)and another EBV-positive epithelial hybrid cell line (A2L/AH), the effect of host cell regulation and human β-interferon (IFN-β) treatment on the induction of EBV-related antigens or cell proliferation was examined.Generally, KB / P3 cells proliferated more rapidly than A2L /AH cells, demonstrating EBV-associated nuclear antigen (EBNA ) in more than 90 % of the cells. The treatment of KB/P3 and A2LAH cells with 5-iodo-2-deoxyuridine (IUdR ) or sodium n-butyrate (SnB) induced both early antigens (EA) and viral capsid antigens (VCA ), while only EA without VCA was induced in the case of treatment of non-producer lymphoblastoid cells, A2L. Incubation at sub-optimal temperature (32°C) also induced both EA and VCA in KB/P3 cells, but not in A2L/AH cells. These results suggest that some factor (s ) produced in KB-AH cells which were responsible for depression of a complete virus productive cycle in KB/P3 cells might not function when treated with these chemical inducers or under sub-optimal incubation conditions.When both hybrid cell lines were subjected to the action of IFN-β for a long time (over two weeks ), cell proliferation was suppressed in proportion to the period of contact and to the concentration of IFN-β. Induction of EBV-related antigens (EA and VCA) in KB/P3 cells by SnB similarly decreased after pre-treatment of the cells with IFN-β for a long time. Combined IFN-β treatment of the cells with 5-FU (5-fluouracil ), but not with MMC (mitomycin -C ), for a long time had additive effects on the anti-proliferative and anti-viral actions of anti-cancer drug. These results may be promising evidence of the possible clinical application of IFN and anti-cancer drugs in the treatment of nasopharyngeal carcinoma (NPC), as EBV-related carcinogenesis is strongly implicated in NPC.

A monoclonal antibody against a Burkitt lymphoma associated antigen has an anti‐Pk red blood cell specificity

The blood group specificity of a rat IgM monoclonal antibody (38-13) directed against most EBV-positive or -negative Burkitt's lymphoma was investigated. The target antigen was previously identified as the neutral glycolipid ceramide trihexoside (CTH), a substance which accumulates in red cells from very rare individuals of the Pk phenotype and which appears as a normal intermediate in the biosynthetic pathway of the P blood group antigen (globoside). The 38-13 antibody agglutinated Pk1 and Pk2 red cells at 4 degrees C with a very high titre but was inactive against native P1, P2 and p erythrocytes, although a very weak activity was noticed towards papain-treated P1 and P2 erythrocytes. These results were confirmed by an indirect radio-binding assay which also demonstrated that the 38-13 antibody reacted with lymphocytes, fibroblasts and EBV-positive lymphoblastoid cell lines derived from Pk1 and Pk2 individuals but not from other donors. These findings demonstrate that the 38-13 monoclonal antibody previously considered as specific of Burkitt cells could be routinely used as an anti-Pk blood group typing reagent. The mechanism of CTH accumulation in Burkitt cells and Pk red cells is probably different and might be associated respectively with the activation of an alpha-4-galactosyltransferase or the genetic blockage of a beta-3-N-acetylgalactosaminyltransferase.

Structure of the Epstein-Barr virus nuclear antigen as probed with monoclonal antibodies

Five hybridoma cell lines secreting antibodies directed against an Epstein-Barr virus (EBV) nuclear antigen ( BamHI K antigen) have been isolated. All five antibodies detect this antigen in a variety of EBV-positive lymphoblastoid cell lines. The reaction of these antibodies with several mutant forms of this protein has allowed the antibody binding site(s) to be predicted.

Antibodies against a synthetic peptide identify the Epstein-Barr virus-determined nuclear antigen.

Five peptides corresponding to amino acid sequences predicted from all three reading frames of the nucleotide sequence of the third internal repeat array (IR3) of the Epstein-Barr virus (EBV) genome were synthesized chemically. All five peptides elicited antipeptide antibodies in rabbits. The antiserum raised against a 14-residue copolymer of glycine and alanine gave brilliant EBV-specific nuclear staining in the anticomplement immunofluorescence (ACIF) assay, in line with the original definition of the EBV-determined nuclear antigen (EBNA) [Reedman, B. M. & Klein, G. (1973) Int. J. Cancer 11, 499-520]. Eight EBNA and EBV DNA-carrying lines showed nuclear staining with the antipeptide antibody, whereas five EBV DNA negative lines failed to stain. The staining pattern was more discretely punctate than the finely dispersed diffuse EBNA staining obtained with human antisera. Human EBV antibody-positive but not EBV-negative sera reacted with the synthetic peptide in an ELISA test. The peptide-specific antibodies were purified from the sera of healthy EBV-seropositive persons by affinity chromatography with the peptide. They gave an EBV-specific, brilliant punctate nuclear ACIF staining similar to that of the rabbit antipeptide antibodies. It was concluded that the glycine-alanine structure encoded by the IR3 region contains a native determinant of EBNA, detected by the ACIF test. Immunoblotting with the rabbit and human peptide-specific antibodies identified poly-peptides that varied between 70 and 92 kilodaltons in size in different EBV-positive cell lines, corresponding closely to a previously identified variation pattern in the size of EBNA. In addition, rabbit antipeptide antibodies identified two cellular polypeptides, 44 and 49 kilodaltons in size.

Virologic, immunologic, and clinical observations on a patient during the incubation, acute, and convalescent phases of infectious mononucleosis

One patient with infectious mononucleosis (IM) was studied from the probable time of Epstein-Barr virus (EBV) infection (38 days before the onset of clinically overt disease), during the incubation and acute phases, until 6 months after clinical remission. Analysis of spontaneous outgrowth of EBV-carrying lymphoblastoid cells, by limiting dilution on feeder layer cultures, showed that virus containing B lymphocytes are already present early during the incubation period. Also low interferon serum levels were detected early after infection, and only before the onset of clinical disease. All other studied clinical laboratory and virus-associated variables were within normal range during the incubation phase, but changed to a pattern characteristic of IM in parallel to the clinical symptoms. During the acute disease EBV-associated nuclear antigen (EBNA)-positive cells could be directly detected among the lymphocytes, and antibodies to EBV antigens appeared. Lymphocytes stained by monoclonal antibodies, detecting Ia-like determinants (activated cells) and supressor cells, increased dramatically, in parallel to a strong increase of functional suppressor cell activity, measured by inhibition of blastogenesis and PWM-induced immunoglobulin production. During the acute phase there was also a decrease of spontaneous cytotoxicity against the NK-sensitive cell line K562, while cytotoxicity (spontaneous) against an autologous EBV-positive lymphoblastoid cell line (LCL) was detected only during this phase. These reactions correlated to the presence of blasts, and the autologous reaction was exerted mainly by Fc-receptor-negative cells. Lymphokine production in response to EBV antigens was also initiated during the acute phase. During the convalescence period the serological and cellular immune parameters adjusted to the pattern of a normal EBV-seropositive person.

Restrictions upon Epstein-Barr virus infection of the leukemic cell are demonstrated in patients with hairy cell leukemia.

We tested the hypothesis that Epstein-Barr virus (EBV) might actually infect leukemic hairy cells in vivo by examining those cells for the EBV-receptor, EBV nuclear antigen (EBNA) and membrane antigen (MA), for spontaneous transformation and rescue of infectious virus and for presence of EBV genome. EBV-receptors were found on subpopulations of leukemic cells from each of 7 patients with hairy cell leukemia (HCL) tested. MA was present on low numbers (1-5 per cent) of fresh leukemic cells of 7 patients and in some instances occurred with a greater frequency after 3 to 5 days in culture, with or without 12-O-tetradecanoylphorbol-13-acetate. In 11 fresh leukemic cell preparations from 8 HCL patients, no EBNA was demonstrated. However, 2 samples after 4 days in culture expressed low frequencies of EBNA-positive cells. Spontaneous, EBV-positive cell lines were established with a high transformation efficiency from 3 HCL blood samples but not from 8 other specimens. Infectious EBV could be rescued from some hairy leukemic cell preparations by co-cultivation with cord blood lymphocytes. These results demonstrated that leukemic cell populations harbored infectious EBV, that the leukemic cells expressed virus receptors and suggested that a small subpopulation of leukemic cells might become infected in vivo at least transiently and possibly transformed in vitro by EBV. To test for the extent of occult in vivo infection of leukemic cells with EBV, Southern type hybridization studies were performed with a probe for EBV genome (Bam HI W). At a sensitivity level of 0.1 genome per cell, EBV genome was not detected in the leukemic cell populations of 7 patients. We conclude that host defence mechanisms protecting these individuals from EBV also prevent infections of the leukemic cell and/or most hairy leukemic cells are not suitable targets for both infection and transformation.

Diploid human lymphoblastoid and Burkitt lymphoma cell lines: susceptibility to murine NK cells and heterotransplantation to nude mice.

Human lymphoid cell lines which had been classified on the basis of studies on clonality and morphological, on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell line (LCL) of presumed non-neoplastic origin and Burkitt lymphoma (BL) lines of proven malignant origin, were tested for susceptibility to natural killer (NK) cells obtained from the spleens of athymic nude mice. The 20 lines included normal diploid LCL and aneuploid BL lines. All cells carried the Epstein-Barr virus (EBV) genome. In addition, two EBV-negative BL lines were tested. The pronase-induced release of 14C-DNA from 14C-thymidine-labelled target cells was used to assess the sensitivity of the cell lines to NK activity. When attempts were made to correlate the growth of the EBV-positive LCL and the EBV-positive BL cell lines in the subcutaneous space of adult nude mice with their susceptibility to NK cells, no significant correlation was observed. The EBV-negative BL cell line, Ramos, however, could be transplanted subcutaneously in nude mice and was more resistant to NK activity than was the EBV-negative BL cell line, BJAB, which cannot be transplanted subcutaneously. Growth of heterotransplanted EBV-converted cell lines in the subcutaneous space of adult nude mice may be influenced by immune effectors other than NK cells.

Human leukemic “null” cell line (NALL-1)

A human lymphoblast cell line, NALL-1, was established from the peripheral blood of a patient with acute lymphoblastic leukemia (ALL). NALL-1 cells had neither properties of T and B cells nor Epstein-Barr virus (EBV). Many characteristics of the NALL-1 line were distinct from those of numerous EBV-positive lymphoblastoid cell lines previously reported. NALL-1 cells are considered to have originated from the donor's leukemic cells on the basis of their cytogenetic, morphologic and functional features. The NALL-1 line is the first human leukemic "null" cell line derived from ALL. The significance of this cell line is disscussed.