Induction of Epstein-Barr virus-associated early antigen in different lymphoid cell lines with ultraviolet-irradiated P3HR-1 virus.

High-titer stocks of the P3HR-1 variant of Epstein-Barr virus (EBV), which had been purified to remove substantial amounts of uv-absorbing impurities, were exposed to various doses of radiation at 254 nm. The residual early antigen (EA)-inducing potential of the irradiated virus was quantitated on different lymphoid cell lines. Infection of the Raji and Daudi cell lines, which contain multiple copies of the EBV genome in latent form, gave multihit survival curves for expression of EA. In contrast, infection of BJA and Ramos cells, which are two rare human B-cell lines which do not contain detectable amounts of EBV-DNA, gave single-hit viral survival curves. Moreover, the uv dose for 37% survival of the EA-inducing potential of the virus preparation was 23 J m^?2 when tested on the genome-negative cell lines, and 300 J m^?2 when the Raji and Daudi lines were superinfected. The data are consistent with a requirement of an intact EBV genome for EA expression in genome-negative cells, while resident viral genomes appear to function in the rescue of uv-inactivated superinfecting genomes in the EBV-carrying cell lines.     

An EBNA-negative, EBV-genome-positive human lymphoblast cell line in which superinfecting EBV DNA is not maintained

A human B-lymphoid cell line, designated TG8, which does not express detectable levels of the EBV (Epstein-Barr virus)-associated nuclear antigen (EBNA), yet carries an average of one to two plasmid copies of the P3HR-1 EBV genome has been identified. TG8 can be superinfected by B95-8 EBV, resulting in up to 60-70% of the population becoming EBNA-positive and 20-30% of the incoming EBV genomes becoming circular by 48 hr postinfection. Neither EBNA expression nor the superinfecting viral DNA is maintained in the population. It is concluded that (1) superinfection of this EBV-genome-positive lymphoblast cell line leads to detectable EBNA expression and circularization of the incoming viral genome and (2) the incoming viral genome and detectable EBNA expression are selectively lost, whereas the endogenous viral plasmid DNA is maintained.     

Heterogeneity of Epstein-Barr virus. II. Induction of early antigens (EA) by complementation.

Cells of Epstein-Barr virus (EBV)-negative human B lymphoma lines BJA and Ramos were converted into EBV genome carriers by virus isolates from P3HR-1 and B 95-8 cells (Fresen and zur Hausen, 1976). Cloning of P3HR-1 virus-converted BJA cells resulted in clones with two different Epstein-Barr nuclear antigen (EBNA) patterns: a faint granular EBNA staining and clones with a brilliant EBNA expression (Fresen et al., 1977). The latter always segregated EBNA-negative cells from which one EBNA-negative subclone (B1-28) was isolated. Induction of early antigens (EA) was studied by infecting parental lines (BJA and Ramos), converted lines (BJA-HR1K, BJA-B 95-8, Ramos-HR1K, Ramos-B 95-8), the BJA-HR1K clones A5 (faint granular EBNA expression) and B1-19 (brilliant EBNA expression), the EBNA-negative subclone B1-28, and Raji cells with EBV from P3HR-1 and B 95-8 cells, respectively. The following results were obtained: (1) EA induction by P3HR-1 virus is enhanced on the average 14-fold in EBV genome-harboring cells when compared to genome-negative lines. (2) B 95-8 virus induces EA only in P3HR-1 virus-converted cells and to a small extent also in Raji cells. A significant EA induction occurs in the A5 clone of BJA-HR1K, whereas the brilliantly EBNA-expressing B1-19 clone is not induced. B 95-8 virus-converted cells cannot be induced by B 95-8 virus. (3) EA induction following infection of EBV genome-carrying cells is directly proportional to the dilution of the infecting virus. In EBV genome-free cells, EA induction is reduced by the square of the dilution factor. These results imply that resident genomes complement superinfecting genomes in EA induction by EBV and that two different populations of genomes (present in P3HR-1 virus isolates) are required for EA induction following infection of B lymphoblasts.     

Appearance of early and late components of Epstein-Barr virus-associated membrane antigen in Daudi cells superinfected with P3HR-1 virus.

This study investigated the synthesis of membrane antigen (MA) as well as virus capsid antigen (VCA) and early antigen (EA) in Daudi cells which had been superinfected with the P3HR-1 strain of Epstein-Barr virus (EBV) and then treated with trypsin to remove initially absorbed MA-positive material from the cell surface. Synthesis of MA, VCA and EA was completely inhibited by puromycin. A marked reduction in the frequency of MA positive cells was observed in superinfected cells cultured in the presence of either cytosine arabinoside (Ara-C) or phosphonoacetate (PA); however, a small fraction of MA synthesis occurred, suggesting an inhibitor insensitive component in MA, A differential absorption of EBV antibody-positive human serum with the Ara-C treated or untreated infected cells detected two antigenically different components in MA: early (Ara-C insensitive) and late (Ara-C sensitive) MA.     

Contrasting effects of hydroxyurea on cell growth and reduction in Epstein-Barr virus genomes in EBV-infected epithelioid cell lines vs Burkitt's lymphoma cell lines

Eliminating Epstein-Barr virus (EBV) genomes from infected cells is an intriguing theoretical strategy in therapy for EBV-associated malignant diseases. Respective patterns were characterized for hydroxyurea (HU)-promoted loss of EBV genomes from EBV-infected epithelioid cell lines derived from the noncancerous portion of gastric carcinoma tissues and Burkitt's lymphoma (BL) cell lines. Epithelioid cell lines GT38 and PN were less sensitivity to HU than BL cell lines Akata, Raji, and Daudi in terms of cell growth inhibition and cell cycle arrest. On passage in medium with 50 microM HU, the fraction of EBV nuclear antigen (EBNA)-positive cells was reduced substantially in the BL cell lines, but only slightly in the epithelioid cell lines. EBV DNA was reduced in Akata, Raji, and Daudi cells upon passage in 50 microM HU by 95%, 70%, and 50%, respectively, but by only 10% in GT38 cells, in which EBV DNA reduction was enhanced at increased concentrations of HU. This indicates that EBV genome is more easily lost from BL cell lines than from epithelioid cell lines upon culturing in HU. These findings support the view that the elimination of EBV could be therapeutically effective in EBV-associated BL by HU.     

Observations on the resistance of Fpstein-Barr virus DNA synthesis to hydroxyurea

The EBV genome was expressed in EBV “negative” D98/HR-1 somatic cell hybrids following treatment with IUdR as monitored by induction of early antigen (EA), virus capsid antigen (VCA), and virus particles. When the hybrid cells were treated with hydroxyurea (HU) following induction, total DNA synthesis was dramatically reduced but expression of both EA and VCA was not altered. The number of EBV genome equivalents increased following induction with IUdR in the presence of 5 mM HU. Synthesis of EBV specific DNA proceeded to the same degree with or without treatment with HU in lymphoblastoid cells infected with EBV while HU treatment reduced total DNA synthesis by 85–90%. Cytosine arabinoside (ara-C) used in the same manner as HU in IUdR induced hybrid cells dramatically reduced cellular DNA synthesis and totally inhibited EBV DNA synthesis.Of all viruses tested thus far, EBV is unique in that EBV specific DNA synthesis is refractile to inhibition by treatment with HU. Several possible explanations for this unique phenomenon are presented.     

Expression of altered ribonucleotide reductase activity associated with the replication of the Epstein-Barr virus.

The expression of the Epstein-Barr virus (EBV) genome can be regulated in the epithelial/Burkitt hybrid (D98/HR-1) cell line and Raji lymphoblastoid nonproducer cell line by induction with 5-iododeoxyuridine (IUdR) or by superinfection with EBV. Extracts of control and induced D98/HR-1 and Raji cells were assayed for ribonucleotide reductase activity in the presence and absence of hydroxyurea (HU). Enzyme activity of control D98/HR-1 and Raji cells was inhibited by greater than 70%, by HU at both low (2 × 10^?4 M) bot high (5 × 10^?4 M) concentrations; however, the reductase activities of IUdR-induced D98/HR-1 cells, superinfected Raji cells, and IUdR-induced Raji cells were resistant to both levels of HU, and enzyme activities of 85 to +100% of control values were obtained in all cases. Under conditions that allowed only partial expression of the EBV genome (before removal of IUdR; early time after superinfection), very significant levels (70–85°k) of HU-resistant enzyme activity were obtained in the presence of 2 × 10^?4 M HU, whereas only 30 to 45% of control reductase activity was observed at the high HU concentration. Mixing experiments employing combinations of various D98/HR-1 control and induced cell extracts indicated that the HU-resistant reductase activity present in induced 1)98/HR-1 cells was due to the presence of an altered enzyme activity and not due to some nonenzymatic factor(s). Additional experiments, in which HU was preincubated with control and induced cell extracts, showed that the HU-resistant ribonucleotide reductase in extracts of induced D98/HR-1 cells was not due to inactivation of the inhibitor by an enzyme or factor present in these cells. These findings of an altered ribonucleotide reductase activity associated with EBV replication, considered in light of similar findings for equine herpesvirus type 1 and herpes simplex virus types 1 and 2, suggest that alteration of this enzyme activity may be a feature of herpesvirus replication.     

Epstein-Barr virus latent and replicative gene expression in oral hairy leukoplakia

Oral hairy leukoplakia is an epithelial lesion of the tongue associated with productive infection by Epstein-Barr virus (EBV). However, no data concerning the pattern of EBV latent gene expression have been reported, and it remains unresolved whether true latent infection occurs in basal cell layers of oral hairy leukoplakia. We have studied six cases of oral hairy leukoplakia using monoclonal antibody immunohistology for EBV latent--EB nuclear antigen (EBNA) 1, EBNA 2 and latent membrane protein 1 (LMP 1); immediate-early (BZLF1); and replicative (EA, VCA, MA) proteins, and for the EBV-receptor (CD21 antigen). EBV DNA was demonstrated by nucleic acid in situ hybridization. Mid- to upper-zone keratinocytes contained EBV DNA and co-expressed EBNA 1, EBNA 2 (5 of 6 cases), LMP 1, BZLF1 protein, EA, VCA and MA. No EBV genome or gene expression could be demonstrated in basal or parabasal cells. Spinous keratinocytes were labelled by anti-CD21 antibodies HB5 and B2, but did not express the EBV-receptor as defined by reactivity with OKB7. The co-expression of latent and replicative infection-associated antigens is striking, indicating possible functional roles for latent proteins during the productive cycle. Our results suggest that oral hairy leukoplakia is caused by repeated direct infection of upper epithelial cells with virus from saliva or adjacent replicatively infected cells, rather than by a latent EBV infection of basal epithelial cells with a differentiation-dependent switch to productive infection as previously proposed.     

Complexity of EBV homologous DNA in continuous lymphoblastoid cell lines

The complexity of Epstein-Barr Virus (EBV) homologous DNA in 11 EBV-infected lymphoblastoid cell lines which have been passaged for several years in culture was determined by hybridization of lymphoblastoid cell DNA to DNA extracted from EBV purified from HR-1 cells and labeled in vitro. Five of the cell lines analyzed contained no early (EA) or viral capsid (VCA) antigens which have been associated with the replication of EBV. Two other cell lines contained EA but not VCA. Of the seven VCA-negative cell lines, those which contained some early antigen or in which early antigen could be induced with IUDR had 56, 48, and 25 copies per diploid cell genome of more than 90% of the sequences of EBV DNA. Five cell lines which did not contain EA even after induction had 23, 8, 8, 6, and 2 copies per cell of the EBV genome. The data indicate that there is a correlation between the number of copies of EBV DNA in nonpermissive cells and the ability of these cells to express EA. Three of the five EA and VCA negative nonpermissive cell lines contained DNA homologous to more than 90% of the sequences of EBV DNA. The kinetics of hybridization of the DNA of two other nonpermissive cell lines, Namalwa and SKL, which contain two and eight copies, respectively, of some EBV DNA sequences, suggest but do not prove that these cell lines may contain incomplete viral genomes. The retention of the full complexity of viral DNA in most nonpermissive lymphoblastoid cell lines may be related to the relatively large number of copies of the viral genome in these cells.     

Expression of Epstein-Barr virus nuclear antigens 3, 4, and 6 are altered in cell lines containing B-type virus

A high proportion of HIV-positive sera were found to react with 130- and 180-kDa antigens which were present in the Jijoye cell line. The majority of the HIV-positive sera which detected these antigens also contained antibodies to Epstein-Barr virus (EBV) nuclear antigen 2B (EBNA2B) suggesting a relationship between B-type EBV strains and the expression of the 130K/180K antigens. Cell lines were established by infection of B lymphocytes with different A- and B-type strains of EBV. Incubation of these lines with sera from individuals infected with either A-type or B-type EBV strains demonstrated that the 130K and 180K antigens were only expressed by cell lines containing B-type virus. Sera from individuals infected with A-type EBV did not react with the 180K antigen in any cell lines but could detect EBNAs 3, 4, and 6 antigens in the A-type cell lines. Restriction enzyme analysis of the BamHI E region of the EBV genome revealed marked differences between the A and B types of the virus. These results demonstrate that expression of antigens encoded from the BamHI E region of EBV (EBNAs 3, 4, and 6) are altered in cell lines transformed by B-type strains of EBV.     

Persistent infection of BHK21/WI-2 cells with rubella virus and characterization of rubella variants

Summary Persistently infected cell lines of BHK21/WI-2 cells have been established by infection with the wild type rubella virus strain M-33. These cell lines, BHK-MP1 and BHK-MP2, showed immunity-like resistance to superinfection with M-33 virus at both 34° and 39.5° C. They also showed intrinsic interference with the replication of Newcastle Disease Virus at 34° C but not at 39.5° C. They released a small number of infectious virus particles which were temperature sensitive variants, being able to form plaques at 34° C, but not at 39.5° C on BHK21/WI-2 and on its derivative, BSR.When BHK-MP1 cells were cultured at 34° C in growth medium containing 10–20 µg/ml of 5-bromodeoxyuridine (BudR) there was a 5- to 10-fold increase in infectious virus in the medium as compared with the untreated controls. Mitomycin C (0.5 µg/ml) treatment for 7 hours likewise stimulated the release of virus from these cells. The enhancement of viral release by BudR was completely blocked by pretreatment with actinomycin D (5 µg/ml) for 3 hours prior to BudR treatment. Since the variant can be induced by these prophage inducers and inhibited by actinomycin D it is suggested that the viral genome is converted to a DNA provirus which is analogous to the lysogenic state of bacteriophage.With 2 Figures     

Epstein-Barr virus DNA in human lymphoid cell lines: In vitro conversion

Two different nucleic acid hybridization techniques were used to search for Epstein-Barr virus (EBV) DNA in four human lymphoma-derived cell lines of B lymphocyte type: BJAB, Ramos, U-698 M, and U-715 M. These cell lines do not express an EBV-associated nuclear antigen, EBNA. Four stably EBNA-positive sublines, GC-BJAB, AW-Ramos, EHRA-Ramos, and EHRB-Ramos, obtained by exposure of the parent lines BJAB and Ramos to EBV in vitro, were also investigated. The EBNA-negative cell lines did not have detectable amounts of EBV DNA (less than 0.3 EBV genome per cell), while the EBNA-positive sublines contained EBV DNA sequences. However, two of the latter four lines had much less EBV DNA (0.8–2 viral genome equivalents per cell) than typical human lymphoid cell lines.     

The seroepidemiology of infection due to Epstein-Barr virus in southern India

We investigated the seroepidemiology of infection due to Epstein-Barr virus (EBV) in 181 south Indian subjects aged 0-25 years using the indirect immunofluorescence method to titrate antibodies to viral capsid antigen (VCA), nuclear antigen (EBNA), and early antigen (EA). The age-specific prevalence of IgG antibodies to VCA rose rapidly to 90% by the age of 5 years. The prevalence of VCA-specific IgM and the geometric mean titre of VCA-specific IgG antibodies were highest between the ages of 6 months and 2 years, the median age of primary infection being 1.4 years. Thus primary EBV infection occurs early in life. EA antibody prevalence was highest (55%) in the third year of life and remained between 30% and 40% thereafter. This pattern of EA antibody prevalence suggests that the latent EBV infection that persists lifelong after primary infection may be reactivated in many individuals. EBNA antibody prevalence was low until the age of 2 years but rose to 80% in the fourth year. Geometric mean titres of antibodies to EA and EBNA were low and stable at all ages. These results are similar to data from areas where EBV-associated Burkitt's lymphoma is endemic and indicate a high EBV infection load early in life.     

Reversible inhibition by phosphonoacetic acid of human B lymphocyte transformation by Epstein-Barr virus.

B lymphocytes purified by immunoabsorbent chromatography from the peripheral blood lymphocytes of adults provide highly effective targets for infection and transformation by Epstein-Barr virus (EBV). Using this system, the kinetics of DNA synthesis induction due to EBV infection have been characterized. The kinetics show two phases: an early phase, lasting 3 to 4 days, the rate and absolute level of which are dependent upon multiplicity of infection; and a later phase, representing normal exponential growth, the level but not rate of which is dependent upon multiplicity of infection. The induction of DNA synthesis begins at a time (24 hr) which agrees well with the published times for the appearance of the Epstein-Barr virus nuclear antigen (EBNA). Absorption and penetration of the cells by EBV appear to require 1 to 2 hr. The induction of DNA synthesis proceeds normally during the first phase in the presence of phosphonoacetic acid (PAA; 200 μg/ml). Thereafter, DNA synthesis remains at a plateau. Blast-transformed EBNA-positive cells may be isolated from these cultures. Removal of PAA at any time up to 28 days postinfection results in resumed proliferation and outgrowth. These cells express four phenotypic properties of transformation by EBV: (1) induction of DNA synthesis, (2) EBNA expression, (3) blast transformation, and (4) immortalization (survival for at least 28 days). However, they cannot grow out. It is therefore proposed that the phenomenon of EBV infection in the presence of PAA may be termed “abortive transformation.”     

Differential effect of phosphonoacetic acid on early antigen synthesis in two Epstein-Barr virus producer cell lines.

The effect of phosphonoacetic acid (PAA) on the expression of Epstein-Barr virus (EBV)-determined antigens in producer (P3HR-1 and B95-8) lymphoblastoid cell lines (LCLs) and iododeoxyuridine (IUDR)-treated nonproducer cell lines (Raji and F137) was investigated. PAA effectively inhibited spontaneous early antigen (EA) synthesis in the P3HR-1 LCL only, while it had no effect on EA production in the B95-8 cell line nor on the IUDR-induced EA in nonproducer LCLs. The inhibition of spontaneous EA in P3HR-1 cell was dose and time dependant, and removal of PAA from cultures and subsequent propagation of these cells in PAA-free medium did not restore spontaneous EA production. The data reported show that EA is synthesized by at least two mechanisms in P3HR-1 cells, one of which is sensitive to PAA, thus suggesting that viral DNA synthesis is required for spontaneous EA synthesis only in the P3HR-1 cells; however, in these same cells, viral DNA synthesis would not be required for the IUDR-induced EA production.     

Epstein-Barr virus nuclear antigen 1 (EBNA1) induced cytotoxicity in epithelial cells is associated with EBNA1 degradation and processing

Epstein-Barr virus nuclear antigen 1 (EBNA1) has a central role in the maintenance and segregation of the Epstein-Barr virus (EBV) episome and by virtue of a glycine-alanine repeat domain is prevented from being endogenously processed for recognition by HLA class I restricted cytotoxic T lymphocytes (CTLs). We found that EBNA1 expression resulted in growth inhibition and a G2/M arrest in human squamous epithelial cell lines (SCC12F, SVK) but not epithelial cell lines of glandular origin (Hela, Ad/AH). The cytotoxicity of EBNA1 was associated with EBNA1 degradation and both these effects were blocked in SCC12F cells expressing either the anti-apoptotic bcl-2 protein or the EBV homolog of bcl-2, BHRF1. The endogenous degradation of EBNA1 in SVK epithelial cells was associated with specific CTL recognition, an effect not evident in EBNA1-expressing Hela cells. Consistent with the inability of SVK cells to tolerate EBNA1 expression, studies with a recombinant EBV demonstrated that SVK cells are unable to maintain stable virus infection, whereas Hela cells are able to efficiently establish latent EBV infection. These data have important implications for both the cellular requirements necessary to sustain a stable EBV infection and for the possible role of CTL responses in controlling EBV infection of epithelial cells.     

Multiplicity-dependent induction of viral capsid antigen in Raji cells superinfected with Epstein-Barr virus

A quantitative analysis of Epstein-Barr virus (EBV)-induced early antigen (EA) and viral capsid antigen (VCA) syntheses was carried out in Raji cells superinfected with purified, concentrated P3HR-1 EBV. When the cells were exposed to the virus and assessed by immunofluorescence and immunoprecipitation, EA induction occurred significantly (17%) but not VCA (less than 1%), at a low-input multiplicity of infection (MOI) of 10 EBV DNA copies/cell. In contrast, at a high MOI of 500 EBV DNA copies/cell, the majority of cells were positive for both EA (82%) and VCA (61%). The latter VCA synthesis was accompanied by the replication of EBV DNA. Kinetic studies showed that EA induction was directly proportional to the dilution of the infecting virus, while VCA was made following three-hit kinetics. The implications of these results are discussed in relation to the heterogeneous nature of P3HR-1 EBV and a possible role of EA in VCA synthesis.