Polypeptides of the Epstein-Barr virus membrane antigen complex.

Epstein-Barr virus (EBV)-associated membrane antigens have been purified from the plasma membranes of the producer cell line P3HR-1 NONO. The antigens were assayed with a specific rabbit anti-ebv antiserum using an 125I-labeled staphylococcal protein A binding assay. The antigens have been shown to be present on purified plasma membranes. Treatment of the plasma membranes with Triton X-100 allows the separation of two antigenically distinct classes of antigens, one soluble and one insoluble in the detergent. Immunoprecipitates of [125I5- and [35S]methionine-labeled, detergent-soluble antigens contained three major polypeptides of molecular weights of 350,000, 140,000, and 75,003 (on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and several minor components. These polypeptides were all specifically precipitated from four EBV-producer cell lines, P3HR-1, P3HR-1 NONO, B95-8, and 7744. They could not be precipitated from producer cell lines treated with phosphonoacetic acid, which inhibits late viral functions, nor could they be precipitated from nonproducer cell lines. The 350,000 and 75,000 molecular weight polypeptides bound to Ricin and lentil lectin columns; however, most of the 140,000 molecular weight material did not. A component of molecular weight 220,000 (prominent only in P3HR-1 NONO) was probably a degradation product of the 350,000 molecular weight polypeptide.     

Effects of S-adenosylhomocysteine and analogs on Epstein-Barr virus-induced transformation, expression of the Epstein-Barr virus capsid antigen, and methylation of Epstein-Barr virus DNA.

S-Adenosylhomocysteine was found to have no effect on Epstein-Barr virus-induced transformation of B-lymphocytes and to stimulate viral capsid antigen expression only slightly in the FF41-1 cell line. In contrast, the S-adenosylhomocysteine analogs sinefungin and S-isobutyladenosine inhibited Epstein-Barr virus transformation and induced a significant increase in the numbers of cells expressing the viral capsid antigen. An inverse relationship between levels of viral DNA methylation and gene expression was demonstrated.     

Severe thrombocytopenia in Epstein-Barr virus-induced mononucleosis.

Severe thrombocytopenia is a rare complication of Epstein-Barr virus-induced infectious mononucleosis. We evaluated the clinical and laboratory data from seven patients seen between 1976 and 1985 whose lowest platelet counts varied from 3 to 25 x 10(9) per liter. Five of the seven patients were initially thought to have either acute leukemia or idiopathic thrombocytopenic purpura; eventually, however, primary Epstein-Barr virus infections were confirmed in all patients. Two of six patients tested had antiplatelet antibodies during the acute phase of their illnesses. Eight additional patients with acute disease who had only mild thrombocytopenia (94 to 144 x 10(9) per liter) were also tested for platelet antibodies with negative results. Steroid therapy was administered to three patients and platelet transfusions to one. All seven patients recovered with no serious hemorrhagic sequelae.     

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.     

Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen.

Five murine hybridoma lines that produce monoclonal antibodies against Epstein-Barr virus membrane antigen (MA) were established. Immunoprecipitation experiments demonstrated that three of the antibodies precipitated both the 236,000 (236K) MA and the 212K MA. The other two antibodies precipitated the 86K MA. Antibodies against the 236K-212K MA and the 86K MA mediated complement-dependent cytolysis of Epstein-Barr-virus-infected cells. The antibodies against the 86K MA neutralized both the B95-8 and P3HR-1 viruses.     

Rabies virus-induced membrane fusion.

Rabies virus is a member of the rhabdovirus family. It enters cells by a process of receptor mediated endocytosis. Following this step, the viral envelope fuses with the endosomal membrane to allow release of the viral nucleocapsid into the cytoplasm. Fusion is induced by the low pH of the endosomal compartment and is mediated by the single viral glycoprotein G, a homotrimeric integral membrane protein. Rabies virus fusion properties are related to different conformational states of G. By different biochemical and biophysical approaches, it has been demonstrated that G can assume at least three different states: the native (N) state detected at the viral surface above pH 7, the activated (A) hydrophobic state which interacts with the target membrane as a first step of the fusion process, and the fusion inactive (I) conformation. Differently from other fusogenic viruses for which low pH-induced conformational changes are irreversible, there is a pH dependent equilibrium between these states, the equilibrium being shifted toward the I-state at low pH. The objective of this review is to detail recent findings on rhabdovirus-induced membrane fusion and to underline the differences that exist between this viral family and influenza virus which is the best known fusogenic virus. These differences have to be taken into consideration if one wants to have a global understanding of virus-induced membrane fusion.     

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

Biochemical characterization of Epstein-Barr virus membrane antigen associated glycoproteins

A direct fluorination of the imidazole side chain of thyroliberin using a photochemical method is described. Preparation of 5-fluoro-imidazole-thyroliberin and 2-fluoro-imidazole-thyroliberin was achieved by successive diazocoupling, amination and fluorination. Specifically substituted derivatives were purified and identified at each step. 5-fluoro-imidazole-thyroliberin was found as active as TRF on short-term prolactin release in GH₃ cell lines and more active (4.5 times) than the parental peptide on long-term experiments. 2-fluoro-imidazole-thyroliberin biological activity test shall be delayed since its chemical stability is not known precisely.     

Epstein-Barr virus nuclear antigen 2 transactivates latent membrane protein LMP1.

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.     

Demonstration of a surface antigen on Epstein-Barr virus genome-carrying lymphoid cells: distinction from the virus-determined membrane antigen.

A surface antigen (SA) was detected on Epstein-Barr virus (EBV)-carrying lymphoid cell lines by indirect membrane immunofluorescence with an antiserum from a rabbit immunized with Raji cells; the antiserum had been extensively absorbed with normal human blood and tonsil cells. The SA was not detected on normal human umbilical cord and adult peripheral blood lymphocytes or EBV-negative cell lines. Incidences of the SA and EBV-determined membrane antigen (MA) on certain EBV-carrying cell lines were not compatible. Antibody against SA was differentially absorbed by the SA-positive MA-negative cell lines whereas MA antibody was absorbed by MA-positive SA-negative cell lines. The results of cross-absorption tests of antiserum against Raji cells or P3HR-1 cells suggested that SA may contain more than one antigenic determinant.     

Mechanism of Epstein-Barr virus-induced human B-lymphocyte activation

The mechanism of Epstein-Barr virus (EBV) activation of human B lymphocytes toward Ig synthesis was investigated in a direct anti-sheep red blood cell (SRBC) antibody plaque-forming cell (PFC) system. Exposure of human peripheral blood lymphocytes to EBV in vitro resulted in an anti-SRBC PFC response in 12 of 16 normal donors. The EBV-induced anti-SRBC PFC response did not require the presence of autologous helper T lymphocytes, but was inhibited by the presence of autologous concanavalin A-generated suppressor T cells. Live virus was required for B-cell activation since the EBV-induced PFC response was inhibited by exposure of EBV to ultraviolet light. Using fluorescent techniques which detected simultaneous intracytoplasmic (ICP) Ig production and the presence of EB nuclear antigen, we found that most, if not all, EBV-activated ICP Ig-positive cells were virally infected. Thus, these studies suggest that viral infection of Ig-producing B lymphocytes is required for EBV-induced polyclonal B-lymphocyte activation. Although the participation of T lymphocytes is not required for the induction of EBV-triggered B-lymphocyte Ig production, activated T lymphocytes can serve as modulators of this response.     

Phosphorylation of the Epstein-Barr virus nuclear antigen 2.

A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic peptide corresponding to amino acids 464-476 of EBNA-2 as a substrate led to the incorporation of 0.69 mol phosphate/mol peptide indicating that only one of three potential phosphorylation sites within the peptide was modified. The most likely amino acid residues for phosphorylation by CK-2 are Ser469 and Ser470.     

Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production

The major goal of this work is to establish a culture system for the growth of human B lymphocytes at the single-cell level so that the immunoglobulin secreted by the clonal progeny of that cell can be analyzed. A method which involves culturing small numbers (1–1000) of lymphocytes, which have been infected with Epstein-Barr virus (EBV) prior to plating, in round-bottom microtiter plates is described. A feeder layer of irradiated (2500 R) umbilical cord blood lymphocytes to which phytohemagglutinin has been added was found to be optimal. Culture supernatants collected from 3 to 6 weeks postinfection are assayed for the production of IgG and IgM by radioimmunoassay in order to determine the overall cloning efficiency of the system. We have shown that up to 33% of surface Ig-positive cells produce detectable clones in this system. Umbilical cord blood cells are superior to T-cell and macrophage cell lines as feeder layers. Furthermore, culture supernatants from phytohemagglutinin-stimulated umbilical cord lymphocytes do not adequately replace these cells. We also observed that while most IgM-secreting clones continued to produce immunoglobulin during the 7-week time period analyzed, the majority of IgG-secreting clones had a relatively short half-life in vitro. This culture system allows us to examine a significant proportion of the human B-cell population and carry out studies on the frequency of specific antibody- and isotype-producing clones.     

Characterization of Epstein-Barr virus antigens. I. Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen.

The Epstein-Barr virus-soluble (S) antigen extracted from RAJI cells was characterized by sucrose gradient centrifugation, gel filtration, and ion-exchange chromatography. The sedimentation coefficient was estimated to be 8.5S corresponding to a molecular weight of 180,000. The S antigen binds to DEAE-A25 ion exchanger from which it can be eluted with 0.3 M NaCl in Tris buffer (pH 7.2). All fractions which contained complement-fixing S antigen also inhibited the anticomplement immunofluorescence reaction as used to detect the Epstein-Barr virus-associated nuclear antigen. These results are consistent with the hypothesis that the S and Epstein-Barr virus-associated nuclear antigens are either a single antigen or that both activities are present on the same molecule.     

Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors.

Since Epstein-Barr virus (EBV) infection of Burkitt's lymphoma (BL) cells in vitro reproduces many of the activation effects of EBV infection of primary B lymphocytes, mRNAs induced in BL cells have been cloned and identified by subtractive hybridization. Nine genes encode RNAs which are 4- to > 100-fold more abundant after EBV infection. Two of these, the genes for CD21 and vimentin, were previously known to be induced by EBV infection. Five others, the genes for cathepsin H, annexin VI (p68), serglycin proteoglycan core protein, CD44, and the myristylated alanine-rich protein kinase C substrate (MARCKS), are genes which were not previously known to be induced by EBV infection. Two novel genes, EBV-induced genes 1 and 2 (EBI 1 and EBI 2, respectively) can be predicted from their cDNA sequences to encode G protein-coupled peptide receptors. EBI 1 is expressed exclusively in B- and T-lymphocyte cell lines and in lymphoid tissues and is highly homologous to the interleukin 8 receptors. EBI 2 is most closely related to the thrombin receptor. EBI 2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes. EBI 2 is also expressed at lower levels in a promyelocytic and a histiocytic cell line and in pulmonary tissue. These predicted G protein-coupled peptide receptors are more likely to be mediators of EBV effects on B lymphocytes or of normal lymphocyte functions than are genes previously known to be up-regulated by EBV infection.     

Characterization of thymus-derived lymphocyte subsets in acute Epstein-Barr virus-induced infectious mononucleosis.

Changes in thymus-derived (T) lymphocyte subpopulation numbers were studied in patients with acute and convalescent Epstein-Barr virus (EBV)-induced infectious mononucleosis (LM). T cell subsets were characterized by the presence of Fc receptors for IgG (TG), for IgM (TM) or by the absence of either receptor (Tnon-M, non-G). We found that in acute IM, total numbers of T and B lymphocytes were elevated (p less than 0.01). Of the T lymphocyte subsets, the total number of Tnon-M, non-G lymphocytes was increased six fold compared to normal subjects (p less than 0.001) and included the majority of the atypical T lymphocytes. The number of total TG and TM lymphocytes was moderately increased (p less than 0.05). In convalescent IM patients, the number of total T cells remained slightly elevated (p less than 0.02) whereas proportions and absolute numbers of B lymphocytes and T cell subsets returned to near normal levels. Thus, acute Epstein-Barr virus-induced IM is associated with a T lymphocytosis which is composed predominantly of atypical T cells which lack detectable Fc receptors for IgG or IgM.