Functional association of class II antigens with cell surface binding of Epstein-Barr virus

A functional role of class II antigen in the binding of Epstein-Barr virus (EBV) was deduced from the study of membrane proteins on Jijoye, an EBV receptor (EBVR)-positive B cell line, and its mutant, EBVR-negative daughter cell line, P3HR-1. From gel electrophoresis of radiolabeled microsomal membrane proteins and immunoprecipitates, we identified class II antigen on Jijoye but not on P3HR-1 cells and the presence of Ii on both cell lines. The role of these molecules in EBVR function was tested by antibody blocking of virus adsorption. Anti-p23,30 serum (to class II antigen) was found to block binding of EBV to B lymphoblasts under conditions in which normal rabbit serum, rabbit antiserum to butyrate-treated P3HR-1 cells (with ample anti-Ii antibodies), and rabbit anti-p44,12 (to class I antigen and beta 2-microglobulin) serum did not block virus binding. Only one of four commercial monoclonal antibodies (MoAb) to framework epitopes on class II antigens blocked binding of EBV, whereas all four MoAb demonstrated immunofluorescent reactivity with the EBVR+ Raji cells. In previous studies of binding of EBV to hairy leukemic cells, a substantial subpopulation of HLA-DR+, EBVR- cells was identified, in addition to HLA-DR+, EBVR+ cells. These findings were consistent with the view that the HLA-DR complex has a role in the binding of EBV but that other components are also needed for the expression of EBVR function.     

Effect of activation of the Epstein-Barr virus genome on expression of B cell differentiation antigens of Burkitt's lymphoma lines

Using anti-human B cell monoclonal antibodies prepared against B1 (CD20), B2 (CD21), B4 (CD19), and BB-1 (B lymphoblast antigen-1), we compared the expression of B cell differentiation antigens on a Jijoye-P3HR-1 cell line family of Burkitt's lymphomas. The expression of BB-1 and B2 antigens was faint on P3HR-1 K cell line which is an Epstein-Barr virus (EBV) high producer. On the other hand, B1 and B4 antigens were strongly expressed on it. It was also found that BB-1 expression decreased on P3HR-1 cells after activation of intracellular EBV genes by treating chemically with tumor-promoting agent (TPA) and n-butyrate, or on Raji cells on superinfection with EBV. This decrease of BB-1 was blocked by the additional treatment with retinoic acid, an inhibitor of virus replication. Dual immunofluorescence staining analysis showed that the individual cell expressing EBV-associated antigens expressed BB-1 antigen only marginally. The relationship between the change in phenotypes of host B cells and the activation of the EBV genome is discussed.     

Studies on the B lymphoblast antigen No. 1 (BB-1) on a series of Burkitt lymphoma lines differing in the expression of the EBV/C3 receptor complex

In the Jijoye-P3HR-1 family of Burkitt lymphoma sublines, the expression of the B lymphoblast-1 antigen, BB-1, identified by the monoclonal antibody described by Yokochi and colleagues, was found to be strictly related to the expression of the EBV receptor/C3 receptor (EBVR/C3R) complex. It was absent on the receptor-negative P3HR-1 line, present in the original receptor-positive Jijoye line, and reappeared in nonvirus producer sublines derived from P3HR-1 itself. We suggest the BB-1 antigen is related to the EBVR/C3R complex in the Jijoye family, either at the level of genetic or epigenetic determination or at the level of steric interaction on the cell membrane. In all probability, however, the BB-1 antigen is not identical to the receptor itself. It is also clear that a similar relationship does not necessarily apply to other cell lines. In the course of the studies, it was accidentally discovered that propagation of the P3HR-1 cells on newborn instead of fetal calf serum induces the concomitant expression of EBV receptors, C3 receptors, and the BB-1 antigen. The mechanism of this induction is obscure; it does not appear to be related to any significant change in the frequency of virus-producing cells.     

Inhibitory effect of 1-beta-D-arabinofuranosylthymine on synthesis of Epstein-Barr virus

The effect of 1-beta-D-arabinofuranosylthymine (ara-T) on cell growth and synthesis of Epstein-Barr virus (EBV) in human lymphoblastoid cell lines was determined. The growth of P3HR-1 cells was not inhibited by 1 microgram of the drug per ml; however, infectious virus production was strongly inhibited and was accompanied by decreased expression of early antigen (EA) and viral capsid antigen (VCA). The ability of 12-O-tetradecanoylphorbol-13-acetate or n-butyric acid to induce synthesis of VCA, but not EA, in P3HR-1 cells was inhibited by ara-T. Similarly, VCA synthesis but not EA synthesis was inhibited by ara-T in Jijoye cells superinfected with the P3HR-1 strain of EBV. The results suggest that ara-T has a specific inhibitory action against EBV replication.     

Deletion of the Nontransforming Epstein-Barr Virus Strain P3HR-1 Causes Fusion of the Large Internal Repeat to the DSL Region

The nontransforming Epstein-Barr virus (EBV) strain P3HR-1 is known to have a deletion of sequences of the long unique region adjacent to the large internal repeats. The deleted region is believed to be required for initiation of transformation. To establish a more detailed map of the deletion in P3HR-1 virus, SalI-A of the transforming strain M-ABA and of P3HR-1 virus was cloned into the cosmid vector pHC79 and multiplied in Escherichia coli. The cleavage sites for BamHI, BglII, EcoRI, PstI, SacI, SacII, and XhoI were determined in the recombinant plasmid clones. Analysis of the boundary between large internal repeats and the long unique region showed that in M-ABA (EBV) the transition is different from that in B95-8 virus. The map established for SalI-A of P3HR-1 virus revealed that, in contrast to previous reports, the deletion has a size of 6.5 kilobase pairs. It involves the junction between large internal repeats and the long unique region and includes more than half of the rightmost large internal repeat. The site of the deletion in the long unique region is located between a SacI and a SacII site, about 200 base pairs apart from each other. The sequences neighboring the deletion in the long unique region showed homology to the nonrepeated sequences of the DS(R) (duplicated sequence, right) region. Sequences of the large internal repeat are thus fused to sequences of the DS(L) (duplicated sequence, left) region in P3HR-1 virus DNA under elimination of the DS(L) repeats. Jijoye, the parental Burkitt lymphoma cell line from which the P3HR-1 line is derived by single-cell cloning, is known to produce a transforming virus. Analysis of the Jijoye (EBV) genome with cloned M-ABA (EBV) probes specific for the sequences missing in P3HR-1 virus revealed that the sequences of M-ABA (EBV) BamHI-H2 are not represented in Jijoye (EBV). In Jijoye (EBV) the complete DS(L) region including the DS(L) repeats is, however, conserved. Further analysis of Jijoye (EBV) and of Jijoye virustransformed cell lines will be helpful to narrow down the region required for transformation.     

B lymphoblast antigen (BB-1) expressed on Epstein-Barr virus-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt's lymphomas

Two new cell surface antigens expressed on B lymphoblastoid cell lines (B-LCL) were defined with cytotoxic mouse monoclonal antibodies. One marker, BB-1 (for B lymphoblast antigen-1), was detected on human and nonhuman primate B-LCL, Epstein-Barr virus (EBV)-activated B cell blasts, most Burkitt's lymphomas, and Ia+ B lymphoblast-like myelomas. Polyclonal B cell activators such as pokeweed mitogen (PWM) and lipopolysaccharide (LPS) also induced the expression of BB-1 on immunoglobulin (Ig)-positive cells. In contrast, BB-1 could not be detected on normal lymphoid tissues by complement-dependent cytotoxicity and immunofluorescence (IF) assays or by analysis with a fluorescence-activated cell sorter (FACS). T cell blasts, T cell leukemias, and pre-B cell or erythroblastic leukemia cell lines were also BB-1 negative. Of particular interest was the finding that BB-1 was expressed on the Jijoye lymphoma but only marginally on a subline of Jijoye, P3HR-1, that lacks receptors for EBV and produces a defective virus incapable of transforming lymphocytes. A second lymphoblast antigen (LB-1) unlike BB-1, was present on both T and B cell blasts and virus-transformed T- and B-LCL but not on normal lymphoid tissues.     

Kinetics of Ii synthesis, processing, and turnover in n-butyrate-treated Burkitt's lymphoma cell lines which express or do not express class II antigens and in hairy leukemic cells

We have sought to understand the role of the electrophoretically invariant chain (Ii) in class II antigen functions, particularly in certain transformed cells in which we have previously demonstrated hyperexpression of Ii. Molecular structures and relative kinetics of Ii synthesis, processing and turnover were compared in paired, Ia+ and Ia- Burkitt's lymphoma (BL) cell lines and in hairy cell leukemia (HCL) cells. Cells were metabolically labeled with [35S] methionine for 15 min (with or without a cold methionine chase to 3 hr) or were continuously labeled for 3 hr. One- and two-dimensional gel electrophoresis resolved immunoprecipitates formed with a) a heteroantiserum to purified class II antigen (demonstrating alpha and beta chains and Ii associated with that complex), b) a heteroantiserum to hairy cell leukemia (HCL) membranes (demonstrating principally the dominant, basic form of Ii molecules, class I antigens, and some additional proteins), and c) a monoclonal antibody to human Ii. Treatment of Ia+ Jijoye and its daughter, Ia- P3HR-1, BL cells with 4 mM butyrate for 48 hr enhanced the synthesis of the dominant, basic form of Ii but did not affect apparent turnover rates of that pool of Ii chains in either cell line. In Ia+ Jijoye cells but not in Ia- P3HR-1 cells Ii was terminally processed to more acidic, sialic acid-derivatized forms. Butyrate treatment did not alter the relative turnover rate of terminally processed Ii in Jijoye cells. The level of the dominant, basic form of Ii in HCL cells equaled that in butyrate-treated Jijoye cells, and relative turnover rates of this terminally unprocessed Ii pool were similar in HCL and Jijoye cells. However, HCL Ia-associated Ii was not terminally processed, as was Ia-associated Ii in Jijoye cells. The expression of Ia auxiliary proteins, p41 and p25, was also enhanced in Jijoye cells by butyrate treatment and was prominent in HCL cells. From these experiments, we may hypothesize the following. In lymphoblastoid cells, two pathways for Ii turnover could exist. One is through association with Ia complexes and progressive terminal processing of carbohydrate side chains and a second is not associated with Ia or, apparently, with such processing. Because Ii is not found to be terminally processed in the absence of class II antigen, Ia might be considered to direct the processing of a subset of Ii towards some function (rather than vice versa).(ABSTRACT TRUNCATED AT 400 WORDS)     

Non-immortalizing P3J-HR-1 Epstein-Barr virus: a deletion mutant of its transforming parent, Jijoye.

The P3J-HR-1 strain of Epstein-Barr virus (EBV) fails to immortalize human lymphocytes. We wished to understand the nature of the genomic alterations which correlated with the loss of this ability. As a first step, the heterogeneity of DNA molecules in the P3J-HR-1 line was eliminated by cell cloning. Then a physical map was prepared of virion DNA from one cell clone, designated FF452-3. By comparison with the genomes of two EBVs, B95-8 and FF41, which are competent to immortalize lymphocytes, we identified a total of eight modifications of BamHI and EcoRI restriction endonuclease fragments of EBV (FF452-3) DNA consisting of insertions, deletions, or loss of a restriction endonuclease recognition site. To determine which of these alterations might be responsible for the loss of transforming phenotype, we examined homologous DNA fragments of the Jijoye strain of EBV, the progenitor of the HR-1 strain which still retains the ability to immortalize lymphocytes. We also studied viral DNA in lymphocytes transformed in vitro by Jijoye virus. Six of the eight alterations were found both in Jijoye and in clonal HR-1 DNA and were presumably genomic traits characteristic of this lineage of EBV. A small deletion in the BamHI-K fragment of HR-1 DNA was not found in Jijoye virion DNA, but this deletion was present in intracellular Jijoye DNA. Thus only one major genomic lesion in HR-1 DNA, a deletion of at least 2.4 x 10(6) molecular weight of DNA from a fused BamHI-H-Y fragment, consistently distinguished Jijoye DNA from its non-immortalizing P3J-HR-1 derivative. This deletion is likely to affect EBV genes which are directly or indirectly involved in immortalizing lymphocytes.     

A biochemical characterization of feline MHC products: Unusually high expression of class 11 antigens on peripheral blood lymphocytes

The polymorphism of feline MHC antigens was examined using biochemical methods. The following observations were made: (1) feline class I and II antigens are polymorphic. Their biochemical features were established using rabbit and mouse reagents directed against human MHC products; they resemble those observed for other mammalian species; (2) the expression of class II antigens in unstimulated cat peripheral blood lymphocytes (PBLs) appears to be unusually high. Cat PBLs express far more class II than class I antigens, whereas in human Epstein-Barr virus-transformed lines, which are known to express relatively large amounts of class II antigens, the situation is reversed.Abbreviations used in this paper EBV Epstein-Barr virus - FLA feline lymphocyte antigen - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MLR mixed lymphocyte reaction - PBL peripheral blood lymphocyte - RT room temperature - TX-114 Triton X-114 - 1-D IEF one-dimensional isoclectric focusing - 2-D SDS-PAGE twodimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Activation of latent Epstein-Barr virus genomes: selective stimulation of synthesis of chromosomal proteins by a tumor promoter.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a potent inducer of Epstein-Barr virus (EBV) gene expression. The optimal conditions for maximum activation of latent EBV genomes by TPA were determined. Although TPA is able to induce replication of EBV genomes in P3HR-1 cells in all phases of growth, the greatest increase in viral genome copies per cell (15-fold above the control level) occurred in nonproliferating cells as opposed to cells growing exponentially (6-fold above the control level). The synthesis of chromosomal proteins in nonproliferating cells under the conditions that induce maximum activation of latent virus genomes by TPA was studied. Selective stimulation in chromosomal protein synthesis accompanied the increase in EBV genomes in P3HR-1 cells despite an overall reduction in total cellular protein synthesis. Comparison of the chromosomal proteins from TPA-induced P3HR-1 cells and from superinfected Raji cells revealed comigrating chromosomal polypeptides of 145K, 140K, 135K, 110K, 85K, and 55K that are presumably EBV associated. The selective stimulation of synthesis of these chromosomal proteins in TPA-treated P3HR-1 cells was closely associated with the activation of latent EBV genomes.     

Inhibition of heavy chain and beta2-microglobulin synthesis as a mechanism of major histocompatibility complex class I downregulation during Epstein-Barr virus replication

The mechanisms of major histocompatibility complex (MHC) class I downregulation during Epstein-Barr virus (EBV) replication are not well characterized. Here we show that in several cell lines infected with a recombinant EBV strain encoding green fluorescent protein (GFP), the virus lytic cycle coincides with GFP expression, which thus can be used as a marker of virus replication. EBV replication resulted in downregulation of MHC class II and all classical MHC class I alleles independently of viral DNA synthesis or late gene expression. Although assembled MHC class I complexes, the total pool of heavy chains, and beta2-microglobulin (beta2m) were significantly downregulated, free class I heavy chains were stabilized at the surface of cells replicating EBV. Calnexin expression was increased in GFP+ cells, and calnexin and calreticulin accumulated at the cell surface that could contribute to the stabilization of class I heavy chains. Decreased expression levels of another chaperone, ERp57, and TAP2, a transporter associated with antigen processing and presentation, correlated with delayed kinetics of MHC class I maturation. Levels of both class I heavy chain and beta2m mRNA were reduced, and metabolic labeling experiments demonstrated a very low rate of class I heavy chain synthesis in lytically infected cells. MHC class I and MHC class II downregulation was mimicked by pharmacological inhibition of protein synthesis in latently infected cells. Our data suggest that although several mechanisms may contribute to MHC class I downregulation in the course of EBV replication, inhibition of MHC class I synthesis plays the primary role in the process.     

DNA of Epstein-Barr Virus. II. Comparison of the Molecular Weights of Restriction Endonuclease Fragments of the DNA of Epstein-Barr Virus Strains and Identification of End Fragments of the B95-8 Strain

Incubation of the DNA of the B95-8 strain of Epstein-Barr virus [EBV (B95-8) DNA] with EcoRI, Hsu I, Sal I, or Kpn I restriction endonuclease yielded 8 to 15 fragments separable on 0.4% agarose gels and ranging in molecular weight from less than 1 to more than 30 x 10(6). Bam I and Bgl II yielded fragments smaller than 11 x 10(6). Preincubation of EBV (B95-8) DNA with lambda exonuclease resulted in a decrease in the Hsu I A and Sal I A and D fragments, indicating that these fragments are positioned near termini. The electrophoretic profiles of the fragments produced by cleavage of the DNA of the B95-8, HR-1, and Jijoye strains of EBV were each distinctive. The molecular weights of some EcoRI, Hsu I, and Sal I fragments from the DNA of the HR-1 strain of EBV [EBV (HR-1) DNA] and of EcoRI fragments of the DNA of the Jijoye strain of EBV were identical to that of fragments produced by cleavage of EBV (B95-8) DNA with the same enzyme, whereas others were unique to each strain. Some Hsu I, EcoRI, and Sal I fragments of EBV (HR-1) DNA and Kpn I fragments of EBV (B95-8) DNA were present in half-molar abundance relative to the majority of the fragments. In these instances, the sum of the molecular weights of the fragments was in excess of 10(8), the known molecular weight of EBV (HR-1) and (B95-8) DNA. The simplest interpretation of this finding is that each EBV (HR-1), and possibly also (B95-8), DNA preparation contains two populations of DNA molecules that differ in the arrangement of DNA sequences about a single point, such as has been described for herpes simplex virus DNA. Minor fragments could also be observed if there were more than one difference in primary structure of the DNAs. The data do not exclude more extensive heterogeneity in primary structure of the DNA of the HR-1 strain. However, the observation that the relative molar abundance of major and minor fragments of EBV (HR-1) DNA did not vary between preparations from cultures that had been maintained separately for several years favors the former hypothesis over the latter.     

The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.     

Two strains of Epstein-Barr virus (B95-8 and a P3HR-1 subclone) that lack defective genomes induce early antigen and cause abortive infection of Raji cells.

The heterogeneity of Epstein-Barr virus (EBV) obtained from P3HR-1 cells has permitted derivation of a distinct subclone of P3HR-1 (L. Heston, M. Rabson, N. Brown, and G. Miller, Nature (London) 295:160-163, 1982). We have analyzed the biologic properties and genomic structure of this subclonal virus (clone 13) compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV compared with those of parental P3HR-1 and B95-8 viruses. Synthesis of EBV proteins in Raji cells superinfected with virus derived from P3HR-1, clone 13, and B95-8 was analyzed both by fluorography of radiolabeled proteins and by immunoblotting. Highly concentrated preparations of clone 13 and B95-8 virus induced most of the spectrum of EBV proteins in Raji cells with the exception of the 145,000-, 140,000-, and 110,000-molecular-weight proteins, which were either undetectable or reduced. Moreover, both clone 13 and B95-8 viruses also induced the same patterns of early antigen diffuse components as the parental P3HR-1 virus did. However, only P3HR-1 virus could induce EBV DNA synthesis in superinfected Raji cells, as determined both by buoyant density centrifugation and by in situ cytohybridization with biotinylated recombinant EBV DNA probes. Defective heterogeneous molecules present in P3HR-1 virus have been implicated in early antigen induction after superinfection of Raji cells. Therefore, Southern blots of clone 13, P3HR-1, and B95-8 viruses were hybridized to recombinant EBV fragments representing the sequences contained within the defective molecules in P3HR-1. The parental P3HR-1 contained the previously described defective molecules. No evidence for defective molecules was found in clone 13 or B95-8 viruses. These data indicate that concentrated preparations of both clone 13 and B95-8 viruses can induce abortive infection in Raji cells, but while the defective molecules are not needed for induction of early antigen diffuse components, they may be required for the induction of viral DNA synthesis.     

Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus.

The Epstein-Barr virus (EBV) genes expressed in B lymphocytes immortalized in vitro or in Burkitt's lymphoma (BL) cells infected in vivo have been characterized previously; however, the viral products which are essential for immortalization or for establishment of EBV latency are still not known. To approach this question, we compared the kinetics of expression of EBV nuclear antigens and the two EBV-encoded small RNAs, EBER1 and EBER2, after infection of primary B cells or EBV genome-negative BL cells with either an immortalizing EBV strain (B95-8) or the nonimmortalizing deletion mutant (HR-1). Following infection of primary cells with B95-8 virus, EBV nuclear antigen (EBNA)-2 was expressed first, followed by EBNA-1, -3, and -4 (also called leader protein [LP]) and the two small RNAs. Infection of EBV genome-negative BL cells with the same strain of virus resulted in a similar pattern of gene expression, except that the EBNAs appeared together and more rapidly. EBERs were not apparent in one BL cell line converted by B95-8. The only products detected after infection of primary B lymphocytes with the HR-1 deletion mutant were the EBNA-4 (LP) family and trace amounts of EBER1. Although HR-1 could express neither EBNA-1, EBNA-3, nor EBER2 in primary cells, all these products were expressed rapidly after HR-1 infection of EBV genome-negative BL cell lines. The results indicate that the mutation in HR-1 virus affects immortalization not only through failure to express EBNA-2, a gene which is deleted, but also indirectly by curtailing expression of several other EBV genes whose coding regions are intact in the HR-1 virus and normally expressed during latency. The pattern of latent EBV gene expression after HR-1 infection is dependent on the host cell, perhaps through products specific for the cell cycle or the state of B-cell differentiation.     

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.     

Role of oncogenes in the regulation of MHC antigen expression.

Class I and class II major histocompatibility complex (MHC) antigens are required for CD8+ cytotoxic T cells and CD4+ helper T-cells, respectively, to recognize foreign antigen. Regulating the levels of expression of these MHC antigens regulates the T-cell responses [1]. This regulation is mainly carried out by the interferons (IFN), which are produced in the disease state. Type I IFN (IFN alpha or IFN beta; collectively 'IFN alpha beta) up-regulates class I MHC and IFN gamma up-regulates class I and class II MHC. We and others [1-3] have shown that transfection of cells with a variety of oncogenes including ras and myc affects the level of MHC antigen expression. This and other data provide evidence for a scheme in which the signal transduction mechanisms whereby IFN up-regulates MHC antigens involve several (proto) oncogenes.     

Epstein-Barr virus DNA XII. A variable region of the Epstein-Barr virus genome is included in the P3HR-1 deletion.

The P3HR-1 subclone of Jijoye differs from Jijoye and from other Epstein-Barr virus (EBV)-infected cell lines in that the virus produced by P3HR-1 cultures lacks the ability to growth-transform normal B lymphocytes (Heston et al., Nature (London) 295:160-163, 1982; Miller et al., J. Virol. 18:1071-1080, 1976; Miller et al., Proc. Natl. Acad. Sci. U.S.A. 71:4006-4010, 1974; Ragona et al., Virology 101:553-557, 1980). The P3HR-1 virus was known to be deleted for a region which encodes RNA in latently infected, growth-transformed cells (Bornkamm et al., J. Virol. 35:603-618, 1980; Heller et al., J. Virol. 38:632-648, 1981; King et al., J. Virol. 36:506-518, 1980; Raab-Traub et al., J. Virol. 27:388-398, 1978; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934, 1980). This deletion is now more precisely defined. The P3HR-1 genome contains less than 170 base pairs (and possibly none) of the 3,300-base pair U2 region of EBV DNA and is also lacking IR2 (a 123-base pair repeat which is the right boundary of U2). A surprising finding is that EBV isolates vary in part of the U2 region. Two transforming EB viruses, AG876 and Jijoye, are deleted for part of the U2 region including most or all of a fragment, HinfI-c, which encodes part of one of the three more abundant cytoplasmic polyadenylated RNAs of growth-transformed cells (King et al., J. Virol. 36:506-518, 1980; King et al., J. Virol. 38:649-660, 1981; van Santen et al., Proc. Natl. Acad. Sci. U.S.A. 78:1930-1934).     

Inhibition of Epstein-Barr virus (EBV) release from P3HR-1 and B95-8 cell lines by monoclonal antibodies to EBV membrane antigen gp350/220.

Antibody-mediated inhibition of Epstein-Barr virus (EBV) release from the EBV-productive cell lines P3HR-1 and B95-8 was probed with two monoclonal antibodies (MAbs), 72A1 and 2L10, which immunoprecipitated the same EBV membrane antigen (MA) gp350/220 found with the 1B6 MAb with which inhibition of EBV release from P3HR-1 cells was first described. These three MAbs were not equivalent in either MA reactivities or functional effects, reflecting the variable expression of different epitopes of gp350/220. 1B6 recognized MA on P3HR-1 cells, which expressed predominately the gp220 form of MA. 1B6 did not recognize (or barely recognized) a determinant on B95-8 cells. MAbs 2L10 and 72A1 reacted as well with B95-8 cells as they did with P3HR-1 cells. MAbs 1B6 and 2L10 neutralized neither P3HR-1 nor B95-8 virus, but 72A1 neutralized both viruses. MAbs 1B6 and 72A1 inhibited P3HR-1 virus release, as measured by the assay for infectious virus and by DNA hybridization analysis of released virus, but 2L10 had no such activity. 72A1 (but not 1B6) inhibited release of EBV from B95-8 cells. These experiments pointed to the presence of three different epitopes on gp350/220, identified with the respective MAbs and having varying involvement in virus neutralization and virus release inhibition.     

Structure of defective DNA molecules in Epstein-Barr virus preparations from P3HR-1 cells.

Epstein-Barr virus (EBV), isolated from P3HR-1 cells, induces early antigen and viral capsid antigen upon infection of human B-lymphoblasts. The strong early antigen- and viral capsid antigen-inducing activity is only observed in P3HR-1 virus preparations harboring particles with defective genomes, suggesting that this biological activity is directly associated with the defective DNA population. After infection of EBV genome-carrying Raji or EBV genome-negative BJAB cells, defective genomes of P3HR-1 EBV DNA are replicated in excess, depending on the multiplicity of infecting EBV particles. Hybridization of the DNA from such infected cells with 32P-labeled EBV DNA after HindIII cleavage reveals six hypermolar fragments. Mapping of these fragments shows that they form one defective genome unit containing four nonadjacent regions (alpha, beta, gamma, and delta) of the nondefective P3HR-1 EBV DNA. Two of the segments (alpha and beta) contain ca. 17 and 13 megadaltons, respectively, from the terminal regions of the P3HR-1 genome, whereas the two smaller segments (gamma and delta) contain ca. 3.7 and 3.0 megadaltons, respectively, originating from the central portion of the genome. In the defective molecule, the regions gamma and delta are present in the opposite orientation compared with nondefective P3HR-1 EBV DNA. Tandem concatemers are formed by fusion of the alpha and beta regions. Our model suggests that tandem concatemers of three defective genome units can be packaged into virions in P3HR-1 cells.