High irradiation sensitivity of epstein-barr-virus (ebv) in lymphoblastoid-cells from patients with ataxia-telangiectasia and ebv genome-positive burkitts-lymphoma

Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines derived from the peripheral blood of patients with ataxia telangiectasia (AT) and EBV genome-positive Burkitt's lymphoma (BL) were tested for expression of EBV-related lytic antigens by means of irradiation. We used 1 Gy in each experiment, according to the results of the P3HR-1 (derived from African BL) cell line. Significantly higher expression of early antigens (EA) and viral capsid antigen (VCA) was demonstrated in lymphoblastoid cell lines derived from both patients with AT and those with EBV genome-positive BL, as compared to those derived from healthy individuals. These results suggested that defective regulating mechanisms on B lymphocytes, responsible for EBV infection, may underlie for the pathogenesis of development of lymphoproliferative diseases both in patients with AT and EBV genome-positive BL.     

Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Re-exposure of human lymphoblastoid cell lines to Epstein-Barr virus

Human lymphoblastoid lines of various origins which harbour Epstein-Barr virus (EBV)-specific nucleic acid were re-exposed to EBV. Following infection, cells of the non-virus-producing lines, Raji and S 95, predominantly synthesized EBV-specific early antigens (EA), whereas only a small percentage of cells revealed viral capsid antigens (VCA). In Raji cells, the number of VCA-producing cells was paralleled by the percentage of virus-specific DNA-synthesizing cells. In S 95 cells, however, viral DNA-synthesizing cells exceeded the number of VCA-producing cells by a factor of more than 10. Induction of EA in Raji cells was dose-dependent and inversely related to cell growth. Irradiation of the virus by ultraviolet light prior to infection led to reduced infectivity. This reduction seemed to follow single-hit kinetics. Raji cells, previously re-exposed to EBV, showed reduced EA induction after re-infection with EBV, as compared to Raji control cells not previously exposed. Of 10 lines which spontaneously synthesize EBV-specific antigens, seven lines proved to be refractory to re-infection, whereas three were as susceptible as the Raji and S 95 controls. From three of the refractory lines infectious virus could be recovered from the culture medium prior to infection. These results permit the following interpretations: (1) the response of human lymphoblastoid cells after re-infection with EBV results from the infecting virus and not from stimulation of endogenous genomes; (2) cells demonstrating EA synthesis ultimately die; (3) re-exposure to EBV increases the resistance to re-infection of the surviving cells; and (4) cell lines producing infectious EBV are refractory to re-infection. It is suggested that the spontaneous synthesis of infectious virus favours the selection of resistant cells.     

Abortive expression of the Epstein-Barr virus (EBV) cycle in a variety of EBV DNA-containing cell lines, as reflected by nucleic acid hybridization in situ.

A variety of Epstein-Barr virus (EBV) DNA-containing cell lines have been tested for the expression of the EBV-associated antigens EBNA (nuclear antigen), EA (early antigen), and VCA (viral capsid antigen), and for the presence of cells containing disproportionate amounts of EBV DNA. The antigen tests utilized immunofluorescence and 125I-labelled antibodies combined with autoradiography. EBV-DNA was detected by in situ hybridization with 3H-labelled EBV RNA complementary to P3HR-1 EBV DNA (P-EBVcRNA). The P-EBVcRNA has been shown to represent the majority of the P3HR-1 EBV DNA sequences. It was concluded that EBV DNA-containing cell lines can be divided into those that express only EBNA, those that express EBNA and EA and those that express EBNA, EA and VCA and also contain cells that undergo disproportionate EBV DNA synthesis. Consequently, in some cell lines there is an abortive expression of the EBV cycle in that some cells spontaneously express EA but fail to continue further to viral DNA synthesis. A similar pattern can be found after experimental induction of the EBV cycle, suggesting that related mechanisms govern the spontaneous expression of the EBV cycle and the extent of its inducibility.     

Immunofluorescence and anti-complement immunofluorescence absorption tests for quantitation of Epstein-Barr virus-associated antigens.

Immunofluorescence absorption methods are described which permit quantitative estimation and differentiation of Epstein-Barr virus (EBV)-associated antigens (virus capsid antigen, VCA, early antigen, EA and EBV-determined nuclear antigen, EBNA) in cell extracts. EBNA was present in all cell lines (producer and non-producer) which carried the EBV-genome, while VCA and EA were present in producer lines only. All the antigens were absent from a lymphoid cell line (MOLT-4) which lacked the EBV-genome, as well as from leukemia cells from peripheral blood. The techniques demonstrated antigenic identity of the various antigens when prepared from different cell lines.     

Segregation of the EBV-determined nuclear antigen (EBNA) in somatic cell hybrids derived from the fusion of a mouse fibroblast and a human Burkitt lymphoma line

Four independently derived hybrids between the mouse fibroblast line A9 and the human, Burkitt-lymphoma-derived lymphoblastoid cell line Daudi were studied for the presence of the Epstein-Barr virus (EBV) genome, the EBV-determined nuclear antigen (EBNA), other EBV-associated antigens, human surface immunoglobulin and the presence of human chromosomes. The four lines differed in the number of their EBV genomes. There was a parallelism between this number, as detected by c/RNA/DNA hybridization, and the frequency of EBNA-positive nuclei. None of the other EBV-antigens, EA, VCA or MA, was expressed at any time, either in the untreated hybrid cells or after IUDR-treatment. The hybrids did not carry detectable surface-associated immunoglobulin or EBV-receptors. The presence of the EBV genome was coincident with the maintenance of human chromosomes, but the hybrids that have lost detectable viral genomes and EBNA still contained a considerable number of human chromosomes, suggesting that the viral genome may be associated with a few chromosomes only.     

Evidence for HTLV-III/LAV expression by primary cultures of T8 cells

The selective targets for HTLV-III/LAV, the causal infectious agent of AIDS and AIDS-related complex (ARC), are T4 cells, apparently because the virus receptor is associated with T4 antigen determinants. This accounts for T4 cell depletion in AIDS and for a decrease of IL-2 production by AIDS peripheral blood lymphocytes (PBL) after in vitro PHA activation. By contrast, T8 cells are not targets for HTLV-III/LAV, since T8 cells from PBL and from long-term cultured T cells (CTC) could not be infected by the virus. We describe 2 samples of PBL from Zairian patients with HTLV-III infection in which HTLV-III was expressed by T8 cells. Evidence that T8 cells were expressing virus was obtained by complement cytotoxicity experiments performed in the presence of OKT8 monoclonal antibody (MAb), which removed HTLV-III-positive cells from cultured T cells producing the virus, and by double labelling experiments, in which some cells exhibit both T8 antigens detected either by IFA (rhodamine) or by rosetting in presence of OKT8 MAbs and HTLV-III antigens detected by IFA (fluorescein) with of anti-HTLV-III p24 and p15 MAbs. Since normal T cells have previously been shown to undergo antigenic diversity, we think these results can be explained by HTLV-III infection of T4 cells which later lost T4 antigens and acquired the T8 phenotype.     

Somatic cell hybrids between human lymphoma cell lines. V. IdUrd inducibility and P3HR-1 superinfectability of Daudi/HeLa (DAD) and Daudi/P3HR-1 (DIP-1) cell lines.

We have studied two types of somatic cell hybrid with regard to expression of the Epstein-Barr virus (EBV) cycle and its regulation. The first, DIP-1, a hybrid formed between two human lymphoma EBV producers (Daudi and P3HR-1), contained EBV DNA, expressed the virus-determined nuclear antigen (EBNA), andwas a producer of the EBV-associated antigens EA (early antigen) and VCA (viral capsid antigen). The second, DAD, a hybrid series of clones formed between Daudi and a HeLa cell derivative (D98), differed with regard to the expression of EBNA, EA, VCA and the content of EBV DNA. EA was regularly induced in the EBV DNA-containing hybrids following treatment with iododeoxyuridine (IdUrd). This induction was greater in lines spontaneously expressing EA. In two hybrids, DIP-1 and DAD10, VCA and virus DNA synthesis were also induced in the presence of IdUrd, the latter being detected by in situ hybridization with P3HR-1 EBV complementary RNA. Finally, while DIP-1 was superinfectable by the P3HR-1 EBV strain, the DAD series of hybrids were refractory to P3HR-1 superinfection and lacked EBV receptors.     

Transformation of lymphocytes by Herpesvirus papio.

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.     

Differential effect of TPA and n-butyrate on induction of Ii and EBV antigens in the P3HR-1 lymphoblastoid cell line

The aim of this study was to test whether EBV induction by TPA or n-butyrate was related directly to hyperexpression of Ii, an electrophoretically invariant, 35 000 dalton, HLA-DR antigen-associated glycoprotein which is abundantly detected in EBV freshly transformed cells and is enhanced by EBV superinfection of lymphoblastoid cell lines. P3HR-1 lymphoblasts were treated with n-butyrate or TPA in variable doses and durations. The augmented expression of Ii, EBV antigens (EA and VCA), DNA synthesis, and cell growth and viability were monitored. n-Butyrate induced hyperexpression of Ii at 2 days with a maximal effective dose of 4 mM, induced EBV antigens (EA and VCA) in 36 per cent of the cells at 2 days, inhibited DNA synthesis and cell growth, and was not cytolytic at 48 h when Ii induction was maximal. TPA did not induce hyperexpression of Ii, induced EBV antigens (EA) in 30 per cent of the cells at 4 days, did not inhibit DNA synthesis and cell growth, and was not cytolytic in the time course and doses studied. Ii expression, therefore, did not appear to be an obligatory consequence of EBV antigen induction. Ii induction might be related to an effect of EBV inducers on cellular DNA synthesis, or on control of the cell cycle, or directly upon Ii gene regulation.     

Increased incidence of IgA antibodies to the Epstein-Barr virus-associated viral capsid antigen and early antigens in patients with chronic lymphocytic leukemia

Antibody titers to Epstein-Barr virus (EBV)-associated early antigens (EA) and the viral capsid antigen (VCA) were determined by ELISA on 263 sera obtained from healthy donors, patients with Hodgkin's disease (HD), non-Hodgkin lymphomas (NHL), infectious mononucleosis (IM), Burkitt's lymphoma (BL), and nasopharyngeal carcinoma (NPC). As expected, most lymphoma patients showed markedly elevated anti-VCA IgG and anti-EA IgG antibody titers. Only one patient in the NHL group (n = 56) consisting of patients with lymphomas other than chronic lymphocytic leukemia (CLL) and hairy-cell leukemia (HCL), and 3 patients with HCL (n = 19) had high antibody titers of the IgA class to VCA and EA. Seventeen out of 48 patients (36%) with CLL had high IgA anti-VCA titers and 10 of these sera (21%) also contained IgA anti-EA. The geometric mean titer (GMT) of IgA anti-VCA was 2,510, the GMT of IgA anti-EA was 780. These antibody titers were about 10 times lower than the corresponding GMT of the NPC patients investigated in this study. The elevated IgG and IgA antibody titers to VCA and EA in CLL and HCL patients seem to reflect an immunodeficiency secondary to the malignant disease leading to reactivation of latent EBV infection. The possibility that at least some of these B-cell lymphomas are associated with EBV cannot be excluded.     

Induction of Epstein-Barr virus in B-lymphoblastoid cells by human immunodeficiency virus type 1

Individuals infected with the human immunodeficiency virus (HIV), the etiologic agent of acquired immunodeficiency syndrome (AIDS), often show symptoms associated with reactivation of Epstein-Barr virus (EBV). In this study, we show that exposure of EBV-positive B lymphocytes to HIV-1 in vitro induced the EBV replicative cycle in these cells, as evidenced by an increased proportion of cells expressing EBV early antigens (EA) and capsid antigens (VCA). Reactivation of EBV by HIV-1 appeared to be virus-dose-dependent and required virus penetration and expression in B cells. Although HIV-1 RNA was detected by in situ hybridization in the majority of HIV-1-infected B lymphocytes, induction of EA and VCA was transient and limited to less than 20% of the cell population. The tumor-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and HIV-1 acted synergistically and had similar kinetics in inducing the expression of EBV. Direct reactivation of EBV by HIV-1 may contribute to the role of EBV as a factor in the genesis of AIDS-related conditions.     

Inducibility of the Epstein-Barr virus (EBV) cycle and surface marker properties of EBV-negative lymphoma lines and their in vitro EBV-converted sublines.

Two EBV-negative lymphoma lines of human B-cell origin, BJAB and Ramos, were compared with altogether six of their in vitro EBV-converted, EBNA- and EBV-DNA-carrying sublines (four of Ramos and two of BJAB derivation). All converted lines closely resembled the parental line with regard to karyotype and HL-A and B antigen typing. Induction of EBV antigens (EA and VCA) by P3HR-1 virus superinfection was either similar in the converted and the negative lines, or somewhat increased in certain converted lines. These findings argue against a simple, virally determined repressor model and emphasize the role or cellular controls in restricting the EBV cycle in virus-carrying B-lymphocyte lines of human origin. IUdR inducibility varied in the different converted lines. There was a possible relationship between average number of EBV-genome equivalents per cell and inducibility. Converted sublines did not differ from the original negative lines with regard to surface immunoglobulin and Fc receptors. There was a dramatic increase in complement-consuming ability, however, following EBV conversion. Among the EBV-positive lines, there was a linear relationship between complement-consuming and EBV-receptor activity, the latter measured by a quantitative absorption test.     

Epstein-Barr virus-related serology in marrow transplant recipients

Serial sera from 50 marrow transplant recipients were examined for their spectra and titers of antibodies to EBV-specific antigens. Immediately before or after transplant, blood products passively transferred antibodies to EB viral capsid antigen (VCA) and EBV nuclear antigen (EBNA). In most recipients, passively-transferred antibodies were replaced by endogenous antibodies regardless of whether donor or recipient had EBV antibodies before transplantation. Commencement or resumption of endogenous EBV antibody production was not associated with signs of infectious mononucleosis or heterophil antibody responses. Antibodies to VCA rose to abnormally high titers, followed successively by antibody to early antigens (EA), and disproportionately low levels of anti-EBNA. Unusually high anti-VCA and anti-EA levels persisted when tests of immune function returned to normal. Antibodies to other herpes group viruses showed no consistent changes. We conclude that (1) EBV does not cause significant clinical problems in marrow transplant recipients; (2) persistent EBV infection can become established or reestablished in the presence of antibodies to EBV; (3) marrow transplant recipients show the same exaggerated immune response to EBV as other immunodeficient patients; and (4) the pattern of EBV-specific antibodies may be a more sensitive measure of defective cell-mediated immunity than most conventional tests of immune function.     

Ultraviolet inactivation of Epstein-Barr virus: effect on synthesis of virus-associated antigens.

The relative sensitivity to ultraviolet (UV) light of genome functions of the P3HR-1 strain of Epstein-Barr virus (EBV) was studied. The formation of viral capsid antigen (VCA) appeared to be more sensitive than that of early antigen (EA), while the synthesis of membrane antigen (MA) was most resistant, as seen on examination in the presence of cytosine arabinoside (Ara-C). However, the appearance of both VCA and EA, but not that of MA, was delayed with UV-irradiated virus, in either the presence or absence of Ara-C. The synthesis of EA and VCA induced by UV-irradiated virus was suppressed in the presence of Ara-C, while that of MA was not.     

Selective cytotoxicity of AIDS virus infection towards HTLV-I-transformed cell lines

Previously, we reported that cells of the human T-cell lymphotropic virus type I (HTLV-I)-transformed lines MT-2 and MT-4 were extensively killed by infection with AIDS retrovirus HTLV-III. We have investigated this phenomenon more systematically using light and electron microscopy as well as immunofluorescence. The cell lines used in the present studies included 14 of those carrying not only human HTLV-I but also related simian agents and 6 HTLV-I-negative T- and B-cell lines. The results showed that the cytocidal effects occurred in the HTLV-I-transformed cell lines exclusively and were not present in further subcultures. In these cell lines the cytotoxic response was closely correlated with the induction of HTLV-III antigens after virus infection. However, cells of 6 HTLV-I-free lines were not killed to a marked extent by HTLV-III and were passaged as continuous producers of AIDS virus. Only 2 cell lines were resistant to the cytocidal effect of HTLV-III among 14 HTLV-I carrying cell lines. They were also resistant to the replication of infected HTLV-III. This AIDS virus-specific cytotoxic effect observed in HTLV-I-transformed cell lines did not appear to be associated with gene expression of the gag and pXs region of HTLV-I genomes. This result may indicate that HTLV-III specifically interferes with some steps of HTLV-I transformation.     

Recovery of transforming EBV from non-producer cells after superinfection with non-transforming P3HR-1 EBV.

Cells of the Raji and NC37 lines can be induced by chemical inducers, such as BrdUrd and IdUrd, or the tumor-promoter TPA to EA-expression only, but do not reveal any VCA synthesis. After superinfection by nontransforming P3HR-1 EBV, however, a varying percentage of the cell population shows VCA synthesis and releases infectious viral particles. The recovered virus differs biologically from P3HR-1 EBV since it transforms human umbilical cord blood lymphocytes into EBNA-positive lymphoblastoid cell lines. Cells of these established lines are susceptible to renewed infection by P3HR-1 EBV which results in EA induction and VCA synthesis. Only cells of one line, NC37-R1, spontaneously produce VCA and EBV particles, which reveal transforming properties and do not induce EA upon superinfection of Raji cells. Infection of P3HR-1 EBV-converted BJA-B cells also leads to EA and VCA induction and the release of viral particles. In contrast to particles recovered from Raji and NC37 cells, no transforming activity was detectable in these virus preparations. According to these data, we propose that viral genomes persisting within Raji and NC37 cells are defective and become complemented by the superinfecting P3HR-1 virus.     

Productive Epstein-Barr viral infection of the human lymphoblastoid cell line 6410 with release of early antigen inducing and transforming virus.

The human lymphoblastoid cell line 6410 was superinfected with P3HR-1 derived Epstein-Barr virus. Subsequent to the first cycle of infection, characterized by early (EA) and virus capsid (VCA) antigen synthesis, the culture, designated 6410-EBV, continued to synthesize VCA. Further immunofluorescence and electron microscopic examination over 18-24 months showed the 6410-EBV culture to be productively infected with EBV. Virus was recovered, in quantity, from the culture fluids and assayed for ability to induce EA synthesis in Raji cells and to transform human umbilical cord lymphocytes. The virus was found to possess both properties, in contrast to P3HR-1 virus which only induces EA. HLA and karyologic analyses of P3HR-1, 6410 and 6410-EBV cultures showed relatedness of 6410-EBV to 6410 cells and dissimilarity to P3HR-1. The biologic activity data coupled with the cytologic analyses confirm a productive infection of the target cells. The reason for differences in biologic activity between the infecting (P3HR-1) and progeny virus is unresolved. It may be speculated that the endogenous EBV genome of 6410 cells was activated and gave rise to a different strain of EBV or to a mixed progeny-parent population as a result of dual infection. Alternatively, the parent strain of virus may have been modfied as a result of interaction with the new host cell.     

Differential induction of Epstein-Barr virus-related antigens in heterokaryon cultures.

Different paterns of induction of Epstein-Barr virus (EBV)-related antigens were observed in heterokaryons produced by Sendai virus-mediated fusion of producer and non-producer human lymphoblastoid cells with various other cell types. EBV-related early antigens (EA) and viral capsid antigen (VCA) could obviously be induced in heterokaryons between producer cells (P3HR-1 and QIMR-WIL), normally expressing these natigens at very low frequency, and human FL or HeLa cells. Positive cells were detected as early as 3 h after fusion and there often followed a rapid increase in positive cells. In contrast, in heterokaryons between non-producer cells (Raji and NC-37) and FL or HeLa cells, only EA but not VCA was induced. EA induction was also evident in fusion of human lymphoblastoid cells with monkey cells (Vero) but with mouse cells (L-M(TK-) C11D and MCB-2) no EBV induction occurred. The EBV induction in heterokaryons was significantly enhanced by 5-iododeoxyuridine treatment.     

Epstein-barr-virus hypersensitivity of lymphocytes from patients with ataxia-telangiectasia

Ataxia telangiectasia (AT), an autosomal recessive disorder with a high incidence of lymphoreticular malignancies including Epstein-Barr virus (EBV)-induced lymphoproliferative disorders (LPD), was investigated to assess the susceptibility to EBV infection and oncogenesis. When the patients' lymphocytes were infected with B95-8 EBV, there was a tendency toward an enhanced growth in semisolid agar, as compared with the healthy donor counterparts. Among the preparations tested, from 14 patients, 2 cell lines showed extremely high colony forming efficiency. The lymphocytes from patients with AT did not contain a large number of EBV target cells, as determined by the maximum frequency of EBV-determined nuclear antigen (EBNA) induction prior to cellular DNA synthesis. Fourteen different lymphoblastoid cell lines derived from the 14 patients with AT were then examined for their EBV inducibility and superinfectibility. By treatment with 12-O-tetradecanoyl-phorbol-13-acetate TPA) and culturing at a lower temperature of 33-degrees-C, early antigen (EA) induction occurred approximately 6-fold and 5-fold higher, respectively, as compared with the lymphoblastoid cell lines derived from healthy controls. Viral capsid antigen (VCA) was also induced significantly by TPA or culturing at lower temperature in the lines from patients with AT, but only slightly in the control counterparts. When the lymphoblastoid cells from patients with AT were exposed to P3HR-1 EBV, EA and VCA syntheses were approximately 6- and 12-fold higher, respectively, than those in the cells derived from the healthy controls. This evidence suggested B lymphocytes of patients with AT were highly susceptible to EBV infection and possibly linked to the development of EBV-induced LPD.