Membrane antigen expression in Epstein-Barr virus-infected Raji cells in the presence of phosphonoacetic acid

The expression of the Epstein-Barr virus (EBV)-induced membrane antigen (MA) in Raji cells experimentally infected with EBV concentrates was inhibited by phosphonoacetic acid (PAA) as determined by membrane immunofluorescence and inhibition of antibody-dependent lymphocyte cytotoxicity. PAA was only effective if present during the first 24 hr following virus adsorption, indicating that the synthesis of MA was primarily a late viral gene function requiring viral DNA synthesis.     

Induction of Epstein-Barr virus early antigens by intercalating chemicals in B95-8 cells

Various intercalating chemicals, such as ethidium bromide (ETB), proflavin (PF), acridine orange (AO), actinomycin D (AMD), methyl green (MG), Hoechst dye (HD), and trioxsalen (TX), have been tested for their effect on the induction of Epstein-Barr virus (EBV) antigens in several lymphoblastoid cell lines. ETB, PF, AD, and AMD enhanced induction of early antigens (EA) in B95-8 cells 3- to 10-fold. In contrast, viral capsid antigen (VCA) synthesis was not significantly affected by this treatment. Besides B958 cells only cells of the M-ABA line were induced to EA synthesis though to a lesser extent. All other cell lines tested did not respond to intercalating chemicals. Simultaneous treatment of B95-8 cells with ETB and 13-cis-retinoic acid inhibits the EA induction efficiently. ETB “superinduced” EA neither in 12-0-tetradecanoyl-phorbol-12-acetate (TPA)- nor in iododeoxyuridine (IUdR)-treated B95-8 cells.     

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.”     

Dissociation between cellular DNA- and histone synthesis following infection by cytomegalovirus in the presence of phosphonoacetic acid

Summary Infection of serum-starved human fibroblasts by human cytomegalovirus in the presence of phosphonoacetic acid was found to induce host cell DNA replication without coordinated histone synthesis.With 2 Figures     

Failure of Epstein-Barr virus-specific cytotoxic T lymphocytes to lyse B cells transformed with the B95-8 strain is mapped to an epitope that associates with the HLA-B8 antigen.

There are two types, A and B, of Epstein-Barr virus (EBV) and B95-8 represents the common type A laboratory strain. Herein, we show in a family study that paternal EBV-specific cytotoxic T lymphocytes (CTL) generated in short-term cultures following stimulation with the autologous B95-8-transformed lymphoblastoid cell line (LCL) or B cells freshly infected with the B95-8 isolate did not lyse haploidentical B95-8 LCL expressing the HLA-A1, -B8, -DR3 paternal haplotype. In contrast, the haploidentical B95-8 LCL expressing the HLA-A11, -B51, -DR7 paternal haplotype was strongly lysed. Moreover, paternal CTL generated in response to stimulation with the B95-8 LCL expressing the haploidentical HLA-A1, -B8, -DR3 paternal haplotype included an allogeneic response against the maternal haplotype but no EBV-specific response as shown by the poor lysis of the autologous LCL target cells. However, stimulation with the haploidentical HLA-A11, -B51, -DR7 paternal haplotype resulted in the generation of both an allogeneic and an EBV-specific response. CTL clones were generated from two HLA-B8+ donors in response to stimulation with the autologous type A LCL transformed with wildtype EBV. The clones were cross-reactive for an immunodominant B95-8-associated peptide epitope that interacted with the HLA-B8 allele but failed to lyse B95-8-transformed LCL targets unless the targets were pre-coated with the exogenous peptide. A CTL clone that was initially stimulated with the autologous BL74 LCL lysed the spontaneous autologous LCL and spontaneous LCL from an HLA-B8+ donor, but failed to lyse the B95-8 LCL from that donor. The observed haplotype preference can be explained in terms of sequence variation between the B95-8 and the corresponding wildtype epitope. Our findings may help to clarify the role of EBV in the pathogenesis of primary Sjögren''s syndrome which is closely associated with HLA-B8.     

Sequence and transcription of Raji Epstein-Barr virus DNA spanning the B95-8 deletion region

The DNA sequence of Raji DNA spanning the deletion found in B95-8 cells has been determined. Three open reading frames and a region of homology with the BamHI-H fragment are found within the deletion. The deletion contains a region of 102-bp repeats which is transcribed into an mRNA. The Raji sequence reported here varies slightly from a smaller M-ABA sequence reported previously. This paper completes the sequence of all parts of the wild-type Epstein-Barr virus genome.     

Crosslinked staphylococcal enterotoxin B stimulates CD8+ T cells only in the presence of unlinked costimulator signals.

The superantigen staphylococcal enterotoxin B (SEB) binds to class II MHC expressing cells and subsequently causes selective activation of T cells carrying appropriate T cell receptor (TCR) V beta chains. Apparently SEB acts as a bifunctional molecule by bridging class II MHC structures with the appropriate TCR-V beta chains. This assumption predicts that immobilized SEB ought to stimulate purified, class II MHC negative murine T cells. We show here that immobilized SEB lacks the ability to trigger murine CD8 T cells. Responsiveness obtained at a high T cell concentration is due to contaminating class II MHC-positive lymphocytes. Complementation of the culture system with syngeneic irradiated B cells blasts effectively restores responsiveness. The proliferating cells exhibit SEB specific cytotoxicity and a bias for V beta 8 expression. Since no evidence for leakiness of SEB covalently bound to sephadex beads was obtained, the data imply that immobilized SEB in fact binds to the TCR of T cells expressing the appropriate V beta chains. However, for primary activation additional costimulatory signals are required which can be provided in an unlinked fashion by activated B cells. Resting B cells are activated by immobilized SEB to cells expressing high costimulator activity. As such, the data point out a third function of SEB.     

Effects of phosphonoacetic acid on subacute myeloopticoneuropathy virus in vitro and in vivo.

The effect of phosphonoacetic acid (PAA) in vitro and in vivo on subacute myeloopticoneuropathy (SMON) virus isolated from the spinal fluid of SMON patients was studied. PAA inhibited multiplication of SMON virus in cultures, but it did not show a direct effect on the virus. The drug did not influence the disease when the medication was started from 10 days after infection of suckling mice. However, the drug did elicit a delay in the incubation period.     

Identification of a region of the Epstein-Barr virus (B95-8) genome required for transformation

In a previous study the identification of a region(s) of the Epstein-Barr virus (EBV) genome, which is associated with transformation, was attempted by marker rescue. A transforming EBV was rescued from D98/HR-1 hybrid cells, which contain the nontransforming HR-1 EBV genome, after transfection with specific BamHI and Charon 4A fragments (J. Stoerker and R. Glaser, Proc. Nat. Acad Sci. USA 80, 1726–1729, 1983). In this study, characterization of the EBV DNA in four human lymphoblastoid cell lines (LCL) transformed with rescued virus was performed. It was found that recombination between the transfected fragments, BamHI H,F,X and the Charon 4A fragment (EB-2636) which is equivalent to the BamHI H,F,X region, and the endogenous HR-1 EBV genome in the D98/HR-1 cells took place. This recombination resulted in the formation of transforming EBV. The EBV DNA in the four LCLs are similar to each other and to HR-1 EBV DNA. However, the EBV DNA in all four LCLs also contain the U2 region plus additional sequences of B95-8 DNA. The U2 region is deleted in HR-1 EBV DNA which :is associated with HR-1 cells and the D98/HR-1 hybrid cells. Thus, transforming activity of the HR-1-like viruses rescued from D98/HR-1 cells was concomitant with the recombination of the 0.26–0.36 region of the EBV genome, suggesting that this region is necessary for at least the initiation of transformation.     

地塞米松对SUNE-1和B95-8细胞株中EB病毒BHRF1基因转录水平的影响

目的：以Ｋ５６２细胞为阴性对照,观察ＳＵＮＥ－１和Ｂ９５８两种含ＥＢＶ－ＢＨＲＦ１基因的肿瘤细胞株在２×１０６ｍｏｌ／Ｌ地塞米松（ｄｅｘａｍｅｔｈａｓｏｎｅ,ＤＥＸ）作用１８ｈ后,细胞内ＥＢＶ－ＢＨＲＦ１基因转录水平的改变。方法：细胞ＲＮＡ的提取、ＲＮＡ的点杂交（Ｄｏｔｂｌｏｔ）及反转录ＰＣＲ（ＲＴ－ＰＣＲ）。结果：上述两种肿瘤细胞在２×１０６ｍｏｌ／Ｌ地塞米松作用１８ｈ等诱导细胞凋亡的条件下,细胞内ＥＢＶ－ＢＨＲＦ１基因的转录水平（ｍＲＮＡ）明显提高。结论：本结果初步显示ＥＢＶ－ＢＨＲＦ１基因的转录与宿主细胞抵抗凋亡的关系密切。     

Inhibition of productive replication of Epstein-Barr virus DNA by phosphonoacetic acid.

Phosphonoacetic acid (100 μg/ml) inhibited EBV DNA replication in superinfected Raji cells but had little effect either on replication of latent EBV DNA or on cellular DNA synthesis. This differential effect of the chemical on EBV DNA replication subsequently was used to determine the number of EBV genomes per cell in productive P3 HRI cells.     

DRB,DQA, DQB AND DPB NUCLEOTIDE SEQUENCES OF SANGUINUS OEDIPUS B95-8

We present eight new nucleotide sequences derived from the second exons of class II genes within the major histocompatibility complex of Sanguinus oedipus (cotton-top tamarin). These comprise two DRB alleles (Saoe-DRB3*0504, -DRB*w1203), two DQA1 alleles (Saoe-DQA1*2501, -DQA1*2502), two DQB1 alleles (Saoe-DQB1*2201, -DQB1*2301), one DQB2 allele (Saoe-DQB2*0101) and one DPB1 allele (Saoe-DPB1*0101).     

Efficacy of phosphonoacetic acid on herpes simplex virus infection of sensory ganglia.

Treatment of mice with phosphonoacetic acid markedly reduced the incidence of latent ganglionic infection with herpes simplex virus when administered within 24 h after viral inoculation, but had no effect on an already established ganglionic infection.     

Selective inhibition by phosphonoacetic acid of MDV DNA replication in a lymphoblastoid cell line.

The effect of phosphonoacetic acid (PA), an inhibitor of herpesvirus DNA polymerase, was studied on replication of Marek's disease virus (MDV) DNA in a lymphoid cell line (MSB-1) originally established from an MD tumor.PA had no significant effect on the growth of MSB-1 cells at concentrations of 100 μg/ml and lower. Also, PA had no effect on synthesis of early virus antigens at concentrations up to 400 μg/ml. PA did, however, suppress the synthesis of virus specific DNA, as indicated by a decrease in the number of virus genomes per cell, and inhibited the synthesis of late virus products, as indicated by the lack of virus assembly. Minimal virus DNA was produced in cells treated with up to 400 μ/ml of PA. Removal of PA from MSB-1 cultures and further propagation of the cells in the absence of PA resulted in an increase in the number of virus genomes per cell and synthesis of late virus products.     

Increased CD95/Fas-induced apoptosis of HIV-specific CD8(+) T cells.

Why HIV-specific CD8(+) T cells ultimately fail to clear or control HIV infection is not known. We show here that HIV-specific CD8(+) T cells exhibit increased sensitivity to CD95/Fas-induced apoptosis. This apoptosis is 3-fold higher compared to CMV-specific CD8(+) T cells from the same patients. HIV-specific CD8(+) T cells express the CD45RA(-)CD62L(-) but lack the CD45RA(+)CD62L(-) T cell effector memory (T(EM)) phenotype. This skewing is not found in CMV- and EBV-specific CD8(+) T cells in HIV-infected individuals. CD95/Fas-induced apoptosis is much higher in the CD45RA(-)CD62L(-) T(EM) cells. However, cytotoxicity and IFNgamma production by HIV-specific CD8(+) T cells is not impaired. Our data suggest that the survival and differentiation of HIV-specific CD8(+) T cells may be compromised by CD95/Fas apoptosis induced by FasL-expressing HIV-infected cells.     

CD8- and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis

The role of CD8 T lymphocytes in the immune response to Mycobacterium tuberculosis infection remains enigmatic, with persuasive reports of both cytolytic and noncytolytic (cytokine-mediated) responses to infection. To address the importance of the cytolytic mechanisms, mice with targeted disruptions for CD8 and perforin or with gene mutations in the CD95/ CD95L signaling pathway were exposed to pulmonary infection. All mice tested showed no differences in their ability to contain the growth of infection during the early phase of disease. As the chronic phase of the disease ensued, however, both CD8- and CD95/CD95L-deficient mice gradually lost their ability to limit bacterial growth. This was associated with a tendency toward pyogenic inflammation in the lung. This tendency was not seen in the perforin gene-disrupted mice. In CD8 gene-disrupted mice, the ability to generate interferon-gamma secreting T cells was unimpaired. Although these cells were capable of entering the lung they were unable to influence the increasing bacterial load in this organ.