An Epstein-Barr virus-specific cytotoxic T cell epitope in EBV nuclear antigen 3 (EBNA 3).

Epstein-Barr virus (EBV)-specific CTL clones were isolated that recognized A-type EBV transformants but not B-type transformants. These A-type-specific CTL clones (HLA B8 restricted) were used to screen peptides derived from the EBV nuclear antigens (EBNAs) 2, 3, 4, and 6 as potential CTL epitopes. Of the 76 peptides screened, one sequence from EBNA 3 (residues 329-353) was recognized by A-type-specific CTL clones after absorption onto target cells (either autologous B-type transformants or PHA blasts). This report is the first description of an EBV target epitope recognized by specific CTL clones.

Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development.

There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type 1 and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA 1 as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the down-regulation of latent antigens can be reversed.

Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus.

To examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-alpha and -beta chains from HLA-B8-restricted, CD8+ cytotoxic T cell clones specific for an immunodominant epitope (FLRGRAYGL) in Epstein-Barr virus (EBV) nuclear antigen 3. Herein, we show that identical TCR protein sequences are used by clones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of alpha and beta chain rearrangements suggest that a persistent viral infection can select for a highly focused memory response and indicates a strong bias in gene segment usage and recombination. A novel double-step semiquantitative polymerase chain reaction (PCR) procedure and direct sequencing of amplified TCR cDNA from fresh lymphocytes derived from three HLA-B8 individuals detected transcripts specific for the conserved beta chain in an EBV-seropositive donor but not in two seronegative donors. This report describes an unprecedented degree of conservation in TCR selected in response to a natural persistent infection.

Binding of benzo(a)pyrene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene to histones.

AKR-2B mouse embryo cells were incubated for 24 hr with [3H]benzo(a)pyrene, and the histones were isolated and analyzed using one- and two-dimensional gel electrophoresis and autoradiography. The results revealed that (a) histones H1, H2A, and H3 incorporated significant amounts of label whereas little or no label was associated with histones H2B and H4 and (b) electrophoresis of the histones in the Triton:acid:urea gel system caused labeled histones to have a slower migration than did the corresponding unlabeled histones. Additional studies such as incubation of (+/-)-7 beta,8 alpha-[3H]dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with nuclei resulted in radioactive labeling of histones H1, H2A, H2B, and H3 and of high-mobility-group proteins HMG1 and HMG2. The low levels of label associated with histone H4 in the whole-cell and nuclear studies were further investigated by incubating isolated histones with (+/-)-7 beta,8 alpha-[3H]dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Under these conditions, negligible amounts of radioactivity were associated with H4, while significant labeling of H1, H2A, H2B, and H3 and other nuclear proteins was observed. The results suggest that factors other than the presence of suitable nucleophilic acceptor sites on the histones may be necessary for carcinogen binding.

Epstein-Barr virus-specific cytotoxic T cell response in cardiac transplant recipients.

PBLs from 10 normal seropositive donors, 15 precardiac transplant patients, and 17 postcardiac transplant patients have been assayed for their ability to mount a cytotoxic T cell response to both A- and B-type EBV. Compared with the results obtained with healthy seropositive donors, pre- and posttransplant patients had significantly weaker T cell responses against both A-type and B-type EBV. Analysis of individual patients showed a preferential T cell response to B-type EBV in 4/15 pre- and 6/17 posttransplant patients and a preferential T cell response to A-type EBV in 1/15 pretransplant and 2/17 posttransplant recipients. PBMCs were obtained from patients and analyzed for the presence of A- and B-type EBV using polymerase chain reaction. EBV types detected in the PBMCs of each individual were correlated with their EBV-specific CTL response. The results obtained indicated that the EBV-specific cytotoxic T cell response of these patients matched the EBV types with which they were infected.

Expression of B-type Epstein-Barr virus in HIV-infected patients and cardiac transplant recipients.

In the present study, peripheral blood mononuclear cells (PBMCs) were obtained from HIV+ subjects as well as cardiac transplant recipients, and the presence of A- and B-type Epstein-Barr virus (EBV) was determined using the polymerase chain reaction (PCR). Of the HIV+ patients studied, 24% were found to be infected with A-type EBV, 27% with B-type EBV, and 39% with both A and B virus types. Analysis of PBMCs from cardiac transplant recipients revealed that 39% were infected with A-type EBV, 33% with B-type EBV, and 28% with both EBV types. These results demonstrate a higher prevalence of infection with B-type EBV in HIV+ patients, than had been found previously by an analysis of spontaneously generated lymphoblastoid cell lines. The data indicated that it is not HIV per se which is responsible for the high incidence of B-type EBV in HIV+ individuals.

The role of Epstein-Barr virus subtypes in human immunodeficiency virus-associated lymphoma.

High grade B cell non-Hodgkin's lymphoma (NHL) is an increasingly important problem in individuals infected with the human immunodeficiency virus (HIV). Fifty percent of these tumours harbour the Epstein-Barr virus (EBV) and there is an equal frequency of EBV A and B-subtypes in the tumours. This contrasts with the recent report that only A-type EBV is associated with Hodgkin's disease. Such studies are challenging the traditional models of EBV-associated lymphomagenesis and showing the way for further studies in this field. This article reviews the studies of EBV subtypes in HIV-associated NHL and uses this new knowledge to discuss the role of EBV in lymphomagenesis.

Burkitt's lymphoma cells are resistant to programmed cell death in the presence of the Epstein-Barr virus latent antigen EBNA-4.

Group I Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cells display a surface phenotype characteristic of germinal centre B cells and readily undergo apoptosis in response to a variety of stimuli, including serum deprivation. Activation of EBV latent gene expression has been shown to increase the survival of these tumour cells by blocking programmed cell death. To investigate the nature of this protection, we assessed the function of the EBV latent EBNA-4 gene in a group I lymphoma line, dG75. Group I BL cells induced to undergo apoptosis in response to serum starvation were protected in the presence of EBNA-4 protein. A possible factor underlying this EBNA-4-associated survival was increased expression of the oncoprotein bcl-2, a known repressor of cell death. Together these data suggest that EBNA-4 plays an important role in the regulation of programmed cell death in BL tumour cells.

Detection of A-type and B-type Epstein-Barr virus in throat washings and lymphocytes.

Peripheral blood lymphocytes, obtained from 94 individuals, were assayed for the presence and type of Epstein-Barr virus (EBV) using primers specific for A-type and B-type EBV in the polymerase chain reaction (PCR). Samples from 16 individuals (17%) were negative for EBV sequences. Of the remaining individuals A-type EBV was detected in 35%, B-type in 21%, and both A- and B-type EBV in 27%. Samples of throat washings were also collected from 33 healthy donors and the presence and type of EBV was determined using PCR. EBV was not detected in 12 donors. However, of those who were excreting EBV, A-type EBV was present in 11 donors (52%), B-type in 7 donors (33%), and both A- and B-types in the remaining 3 donors (14%). These results suggest that infection with B-type EBV and coinfections with both A- and B-type EBV are more common than previously thought.

Modulation of vimentin, the CD40 activation antigen and Burkitt's lymphoma antigen (CD77) by the Epstein-Barr virus nuclear antigen EBNA-4.

The growth transformation of human B cells by Epstein-Barr virus (EBV) is controlled by the coordinate expression of 10 latent viral genes. This transforming capacity is believed to be fundamental to the involvement of the virus in human malignancies of B cell origin. EBV-negative Burkitt's lymphoma (dG75) clones stably expressing one of these EBV-coded antigens, EBNA-4, have been established using an episomal-based plasmid. EBNA-4 expression was found to upregulate the cytoskeletal protein vimentin as well as surface expression of the activation antigen CD40. In addition, the presence of EBNA-4 resulted in downregulation of the Burkitt's lymphoma-associated antigen (BLA/CD77). These studies show for the first time that EBNA-4 modulates the expression of several cellular genes implicated in cell-growth transformation.

Substrate specificity studies on a calf thymus nuclear phosphoprotein kinase.

1. The protein kinase activity associated with the phosphoprotein fraction of calf thymus nuclei has been examined for its ability to phosphorylate exogenous phosphoprotein substrates. beta-Casein and phosvitin are both efficient phosphate acceptors. Phosphorylation of alpha s1 and beta-caseins was shown, directly, to occur at threonyl-49 and threonyl-41 respectively. Both threonyl residues occur within the sequence Thr-Glu-Asp. 2. alpha s1-Casein becomes a much better substrate for the kinase when partially dephosphorylated. A similar response is shown by a phosphopeptide containing the alpha s1-casein phosphate cluster and is due to a new phosphorylation site becoming available. Efficient phosphorylation of beta-casein requires that the phosphate cluster (residues 15-19) be intact and results are presented which are consistent with there being a similar requirement for phosphorylation of the site created in alpha s1-casein by partial dephosphorylation. 3. Comparison of genetic variants of beta-casein as phosphate acceptors for the kinase shows that the presence of lysyl residues close to the phosphorylation site markedly depresses the rate of phosphorylation. Maleylation of beta-casein increases the rate of phosphorylation for all of the variants tested, although to varying extents. Treatment of maleylated beta-casein with trypsin to remove the N-terminal phosphopeptide inhibits phosphorylation by the kinase. 4. The structural determinants of beta-casein allowing it to be efficiently phosphorylated by the kinase are concluded to be the presence of a sequence surrounding the phosphorylation site, which is rich in acidic amino acid residues and from which basic residues are absent. The acidic phosphate cluster of beta-casein also promotes phosphorylation either by interacting directly with the enzyme or through its influence on the conformation of beta-casein.

Western blot analysis of antibody specificities in subacute sclerosing panencephalitis: reactivity to measles virus proteins produced in persistently infected cells.

The specificity of serum antibodies from patients with subacute sclerosing panencephalitis (SSPE) and seropositive controls to measles virus proteins produced in acutely and persistently infected human cells was examined by western blot analysis. Sera from both SSPE patients and controls reacted to the H, N, and F1 virus proteins produced in acutely infected AV3 cells. However, while SSPE-derived sera reacted with the same proteins in persistently infected cells (AV3Al/MV), most control sera failed to react with the hemagglutinin protein produced in such cells (Hp). Most sera also reacted poorly with the M protein from either source, and the reactivity to the P protein was variable. Although the exact reason(s) for the different reactivities to the proteins were not determined, differences in antibody concentration did not appear to be responsible. The dramatic differences in the reactivity of SSPE and control sera to the Hp protein suggest that either the protein coevolves in persistent infections or multiple forms of the protein evolve in such infections and SSPE patients develop broad-spectrum humoral immunity as a consequence of exposure to them. Alternatively, over time there may be selective loss of some H-reactive antibody subsets by individuals who contract measles, but do not develop SSPE.

Induction of Epstein-Barr virus nuclear antigens.

Lymphocytes were infected with the QIMR-WIL strain of Epstein-Barr virus, and the induction of Epstein-Barr virus-associated nuclear antigens was determined by using the protein immunoblot. There was a temporal increase in six antigens, with Epstein-Barr nuclear antigen 2 being detected 1 day after infection. The appearance of these antigens was shown to be independent of cellular DNA synthesis.

The prevalence of antibodies to an Epstein-Barr virus-induced polypeptide (EBNA-2) in the sera of rheumatoid arthritic families.

Using the protein immunoblot technique, antibodies to an Epstein-Barr virus-induced 92 kD polypeptide (EBNA-2) were more frequently present in the sera of patients with rheumatoid arthritis and their consanguineous relatives when compared with a control group. No association of anti-EBNA-2 antibody with the HLA-DR antigens was observed.

Characterization of Epstein-Barr virus nuclear antigen(s) in different cell lines by radioimmunoelectrophoresis.

Utilizing radioimmunoelectrophoresis, Epstein-Barr virus nuclear antigens (EBNA) with molecular weights of 67,000 and 70,000 were detected in NC37 cells, while the Raji cell line contained an antigen with a molecular weight of 70,000. Cell lines containing the B-95 strain of Epstein-Barr virus (EBV) expressed antigens with a molecular weight of 73,000. Three different B-lymphocyte cell lines, established by infection with the B-95 virus, also exhibited 73,000 molecular weight EBNA proteins. Cell lines containing the P3HR-1 strain of EBV demonstrated a single antigen with a molecular weight of 72,000. The results indicate that the molecular weight of EBNA is determined by the strain of infecting virus.

Cutting edge: silencing virus-specific cytotoxic T cell-mediated immune recognition by differential splicing: a novel implication of RNA processing for antigen presentation.

Persistent viruses have developed potent strategies to overcome host immune defenses. In particular, viral interference with Ag presentation by HLA class I molecules can effectively impair the host's CTL function. Here we provide evidence for a novel aspect of differential splicing on endogenous processing of a latent viral transcript resulting in dominant protein isoforms from which the CTL determinant has been deleted. Consequently, virus-infected cells expressing these isoforms were poorly recognized by CTLs. Molecular analysis revealed that this splicing significantly reduced expression of the viral transcript encoding the relevant epitope to levels below the threshold required for CTL recognition. The importance of splicing was further reinforced by the observation of efficient CTL recognition of target cells expressing a truncated viral transcript that abolished differential splicing. Thus, differential splicing, which is a common mechanism of gene regulation in many pathogens, may unexpectedly interfere with immune recognition.

The binding of beta-casein to hydroxyapatite: the effect of phosphate content and location.

The elution behaviour of beta-casein from columns of hydroxyapatite has been studied and the effect examined of the enzymic addition of an extra phosphate residue. When the additional phosphate is located near pre-existing phosphate residues stronger binding to hydroxyapatite is observed. When the additional phosphate is remote from the pre-existing phosphates no increase in strength of binding is observed. It is suggested that the clustering of phosphate residues which characterizes alphas- and beta-caseins can be rationalized on the basis that it facilitates co-operative interactions between these phosphates and the Ca phosphate of the casein micelle and/or basic residues within casein polypeptide chains.