Epstein-Barr virus (EBV) receptors, complement receptors, and EBV infectibility of different lymphocyte fractions of human peripheral blood. I. Complement receptor distribution and complement binding by separated lymphocyte subpopulations.

Human peripheral lymphocytes were fractionated into a variety of B-, T-, and O-cell fractions and were characterized with regard to several surface receptors. There was a strong correlation between the frequency of EAC receptor-positive cells and the percentage of complement membrane fluorescence (CMF)-stained cells following exposure to fresh human serum and subsequent staining with an anti-C3 conjugate. CMF staining did not diminish in C4-deficient or hypogammaglobulinemic serum, or in the presence of EDTA or EGTA-Mg²⁺, but was completely negative with C3-depleted normal human serum. In all likelihood, the staining is therefore due to the direct binding of C3 to preformed receptors on the lymphocyte surface. In addition to the surface Ig-positive B-cell fractions, C3 receptors were also detected on part of the O-cell population and on a proportion of the Fc receptor-positive T cells.     

A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro.

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.     

Epstein-Barr virus recombinants from overlapping cosmid fragments.

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Application of flow cytometry to studies on the distribution, persistence, and regeneration rate of Epstein-Barr virus receptors

Flow cytometry has been applied to study the persistence and regeneration rate of Epstein-Barr virus (EBV) receptors and other membrane proteins in normal and tumor cells. EBV receptors were detected in a binding assay utilizing fluorescein isothiocyanate-conjugated virions (FITC-EBV). After stripping receptors from the cell surface with trypsin, binding of FITC-EBV was rapidly regenerated in the lymphoma cell line Loukes, reaching 75% of the control levels by 3 h. Fresh human lymphocytes regenerated receptors at a much slower rate, reaching 50% of control levels by 24 h. The regenerating receptors did not reappear simultaneously on all the cells. Subpopulation of new receptor-positive cells increased gradually during the incubation at 37 degrees C. Reappearance of receptors was inhibited in the absence of protein synthesis. Conversely, receptors persisted in the nontrypsinized cells in the absence of protein synthesis. These results show that certain parameters of membrane receptor turnover, such as the change in receptor distribution within a cell population, receptor persistance, and regeneration rate, can be measured at the single-cell level by flow cytometry.     

Presence of Epstein-Barr virus receptors, but absence of virus penetration, in cells of an Epstein-Barr virus genome-negative human lymphoblastoid T line (Molt 4).

This paper reports a unique type of interaction of Epstein-Barr virus (EBV) with an EBV receptor-positive, genome-negative human lymphoid T cell line (Molt 4), which can be summarized as follows. Although Molt 4 cells express receptors for EBV, they appear to block the penetration of this virus. These observations are derived from combined studies with immunofluorescence and electron microscopy. It is possible that T cell lines bearing receptors for EBV may express such a control on virus penetration.     

Induction of complement receptor expression in cell lines derived from human undifferentiated lymphomas. II. Characterization of the induced complement receptors and demonstration of the simultaneous induction of EBV receptor

We have studied the specificity of complement receptors induced by theophylline in 2 cell lines derived from undifferentiated lymphomas, one of Burkitt's type, and compared it to that of complement receptors in other cell types. Both C3b and C3d receptors were induced. The induced C3b receptor differed from the C3b receptor of mature normal lymphocytes, polymorphonuclear leukocytes and the cells of a nodular lymphoma in 2 respects. Firstly, it bound C3b much less avidly (by a factor of several hundred-fold) and secondly, we were unable to demonstrate C4b binding. EBV receptors were induced at the same time as complement receptors, and permitted the conversion of a greater fraction of cells to EBNA positivity after experimental infection with EBV. The induction of receptors was not associated with a change in the fluidity of the plasma membranes and our data do not favor a different orientation of induced receptors within the membrane as compared to receptors of other cell types--a potential explanation for the different specificities. Our findings are consistent with the possibility that the complement receptors of lymphocyte precursors differ from these of mature lymphocytes.     

The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells

The genome of Epstein-Barr Virus (EBV) and plasmid derivatives of it are among the most efficient extrachromosomal replicons in mammalian cells. The latent origin of plasmid replication (oriP), when supplied with the viral Epstein-Barr Nuclear Antigen 1 (EBNA1) in trans, provides efficient duplication, partitioning and maintenance of plasmids bearing it. In this review, we detail what is known about the viral cis and trans elements required for plasmid replication. In addition, we describe how the cellular factors that EBV usurps are used to complement the functions of the viral constituents. Finally, we propose a model for the sequential assembly of an EBNA1-dependent origin of DNA synthesis into a pre-Replicative Complex (pre-RC), which functions by making use only of cellular enzymatic activities to carry out the replication of the viral plasmid.     

Reducing the complexity of the transforming Epstein-Barr virus genome to 64 kilobase pairs.

Transformation-competent, replication-defective Epstein-Barr virus (EBV) recombinants which are deleted for 18 kbp of DNA encoding the largest EBNA intron and for 58 kbp of DNA between the EBNA1 and LMP1 genes were constructed. These recombinants were made by transfecting three overlapping cosmid-cloned EBV DNA fragments into cells infected with a lytic replication-competent but transformation-defective EBV (P3HR-1 strain) and were identified by clonal transformation of primary B lymphocytes into lymphoblastoid cell lines. One-third of the lymphoblastoid cell lines were infected with recombinants which had both deletions and carried the EBNA2 and EBNA3 genes from the transfected EBV DNA and therefore are composed mostly or entirely from the transfected EBV DNA fragments. The deleted DNA is absent from cells infected with most of these recombinants, as demonstrated by Southern blot and sensitive PCR analyses for eight different sites within the deleted regions. Cell growth and EBNA, LMP, and BZLF1 gene expression in lymphoblastoid cell lines infected with these recombinants are similar to those in cells infected with wild-type EBV recombinants. Together with previous data, these experiments reduce the complexity of the EBV DNA necessary for transformation of primary B lymphocytes to 64 kbp. The approach should be useful for molecular genetic analyses of transforming EBV genes or for the insertion of heterologous fragments into transforming EBV genomes.     

Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells.

Human lymphoblastoid Raji cells, which do not produce virus, supported replication of Epstein-Barr virus (EBV) upon superinfection. Early antigen, viral capsid antigen, and virions were produced in Raji cells superinfected with EBV. Viral DNA replicated under complete inhibition of host cell DNA synthesis to the extent that a few micrograms of EBV DNA were recovered from 107 superinfected Raji cells, corresponding to 5,000 viral genomes/cell. Homology of the synthesized viral DNA to parental EBV DNA was more than 90%. Virions produced by the Raji cells contained a 55S DNA but failed to induce early antigen, viral capsid antigen, and viral DNA synthesis after a second superinfection of Raji cells.     

Establishment of latent Epstein-Barr virus infection and stable episomal maintenance in murine B-cell lines

Epstein-Barr virus (EBV) is a strict human pathogen for which no small animal models exist. Plasmids that contain the EBV plasmid origin of replication, oriP, and express EBV nuclear antigen 1 (EBNA1) are stably maintained extrachromosomally in human cells, whereas these plasmids replicate poorly in rodent cells. However, the ability of oriP and EBNA1 to maintain the entire EBV episome in proliferating rodent cells has not been determined. Expression of the two human B-cell receptors for EBV on the surfaces of murine B cells allows efficient viral entry that leads to the establishment of latent EBV infection and long-term persistence of the viral genome. Latent gene expression in these cells resembles the latency II profile in that EBNA1 and LMP1 can be detected whereas EBNA2 and the EBNA3s are not expressed.     

Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein.

The Eptstein-Barr virus (EBV)-determined nuclear antigen (EBNA) was solubilized from isolate nuclei of two EBV-transformed cell lines- Raji and AW-Ramos, by high-salt treatment. Its DNA-binding properties were studied by DNA-cellulose chromatography and a 51Cr release complement fixation assay. EBNA binds to both double-stranded and single-stranded calf thymus DNA, showing a higher affinity to double-stranded DNA. There was no detectable difference in the DNA binding of EBNA prepared from Raji and AW-Ramos cells.     

Amplification of Epstein-Barr virus (EBV) DNA by superinfection with a strain of EBV derived from nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) from a nasopharyngeal carcinoma (NPC) hybrid cell line (NPC-KT) lacking defective viral DNA molecules superinfected Raji cells and induced EBV early antigens (EA), as did virus from P3HR-1 cells, which contained defective molecules. The EBV polypeptides induced by NPC-KT appeared to be identical to those induced by P3HR-1 virus. The ability of NPC-KT virus to induce EA was enhanced more than 10-fold by treatment of superinfected cells with dimethyl sulfoxide; however, dimethyl sulfoxide treatment did not enhance superinfection by P3HR-1 virus. After infection, DNA synthesis of both the superinfecting NPC-KT virus and the resident Raji viral genome was induced. In addition to amplified Raji EBV episomal DNA, a fused terminal fragment of NPC-KT viral DNA was detected. The detection of fused terminal DNA fragments suggests that the superinfecting virion DNA either circularizes or polymerizes after superinfection and is possibly amplified through circular or concatenated replicative intermediates.     

Biological properties and viral surface antigens of Burkitt lymphoma- and mononucleosis- derived strains of Epstein-Barr virus released from transformed marmoset cells.

Three strains of Epstein-Barr virus (EBV), two from Burkitt lymphoma (BL) and one from infectious mononucleosis (IM) were used to transform separate cultures of the same batch of primary marmoset leukocytes, and the viruses released from the transformants were compared. The three viruses shared properties of the transforming biotype of EBV, namely, stimulation of DNA synthesis and immortalization of cord blood leukocytes, and failure to induce "early antigen" in lymphoblast lines. All viruses produced more virus in transformed marmoset cells than in transformed human cells, as measured by the number of EBV genomes detected by complementary RNA/DNA hybridization, by virus capsid antigen expression, or by released virions and biologically active virus. Reference human sera and sera from primary EBV infections were used to compare the three virus strains in a virus neutralization test based on inhibition of stimulation of DNA synthesis. Specimens taken late in convalescence from patients with mononucleosis and sera from marmosets experimentally infected with virus from a patient with mononucleosis neutralized the homologous virus, as well as the two virus strains isolated from patients with BL. This finding indicates that viral antigens that elicit neutralizing antibodies are shared among the strains. However, in certain sera the neutralizing-antibody titer against one strain was consistently higher than against another strain. Furthermore, sera taken early after onset of IM contained low levels of neutralizing antibody against IM-derived virus, but failed to neutralize BL-derived virus. These latter findings suggest the existence of heterogeneity among surface antigens of EBVs. The results emphasize the biological and antigenic similarity of EBV isolates from BL and IM and do not suggest major subtype variations. It remains to be determined whether antigenic diversity such as described or virus genome variation detectable by other means is epidemiologically significant.     

Contributions of the Epstein-Barr virus EBNA1 protein to gastric carcinoma

Approximately 10% of gastric carcinomas (GC) are comprised of cells latently infected with Epstein-Barr virus (EBV); however, the mechanism by which EBV contributes to the development of this malignancy is unclear. We have investigated the cellular effects of the only EBV nuclear protein expressed in GC, EBNA1, focusing on promyelocytic leukemia (PML) nuclear bodies (NBs), which play important roles in apoptosis, p53 activation, and tumor suppression. AGS GC cells infected with EBV were found to contain fewer PML NBs and less PML protein than the parental EBV-negative AGS cells, and these levels were restored by silencing EBNA1. Conversely, EBNA1 expression was sufficient to induce the loss of PML NBs and proteins in AGS cells. Consistent with PML functions, EBNA1 expression decreased p53 activation and apoptosis in response to DNA damage and resulted in increased cell survival. In addition, EBNA1 mutants unable to bind CK2 kinase or ubiquitin-specific protease 7 had decreased ability to induce PML loss and to interfere with p53 activation. PML levels in EBV-positive and EBV-negative GC biopsy specimens were then compared by immunohistochemistry. Consistent with the results in the AGS cells, EBV-positive tumors had significantly lower PML levels than EBV-negative tumors. The results indicate that EBV infection of GC cells leads to loss of PML NBs through the action of EBNA1, resulting in impaired responses to DNA damage and promotion of cell survival. Therefore, PML disruption by EBNA1 is one mechanism by which EBV may contribute to the development of gastric cancer.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.