Epstein-Barr virus gene expression in malignant lymphomas induced by experimental virus infection of cottontop tamarins.

Inoculation of cottontop tamarins with a large dose of Epstein-Barr virus (EBV) leads to the induction of multiple EBV genome-positive lymphomas. These tumors have been characterized as oligoclonal or monoclonal large-cell malignant lymphomas that closely resemble the EBV genome-positive B-cell lymphomas that arise in human allograft recipients. The expression of latent and lytic EBV-encoded proteins was investigated in these virus-induced tamarin lymphomas and in derived cell lines. The tamarin tumors were found to express EBV nuclear antigen 1 (EBNA 1), EBNA 2, EBNA leader protein, and the latent membrane protein (LMP) as determined both by immunohistochemical staining and by immunoblotting. However, within the limits of the immunoblotting assays, no expression of the EBNA 3a protein family could be detected. Assays for lytic-cycle proteins by using both polyclonal human sera and monoclonal antibodies against viral capsid antigen, early antigen, and membrane antigen (gp340/220) showed minimal, if any, expression of these antigens in the lymphoma biopsies. In contrast, the cell lines derived from these lymphomas, even in early passage, expressed abundant levels of the lytic-cycle antigens and also expressed the EBNA 3a protein as well as EBNA 1, EBNA 2, EBNA leader protein, and LMP. This finding suggests that the virus-lymphoma cell interaction, in particular the switch to lytic cycle, is subject to some form of host control in vivo. The expression of EBNA 2 and LMP in these tamarin lymphomas strengthens their resemblance to posttransplant lymphomas in humans, since these human tumors are also EBNA 2 and LMP positive (L. S. Young, C. Alfieri, K. Hennessy, H. Evans, C. O'Hara, K. Anderson, A. Rickinson, E. Kieff, and J. I. Cohen, submitted for publication). Since both proteins are known to be important effector molecules of virus-induced B-cell growth transformation in vitro, their expression in these lymphomas constitutes the best evidence for a direct oncogenic role for EBV in vivo.     

Different patterns of Epstein-Barr virus gene expression and of cytotoxic T-cell recognition in B-cell lines infected with transforming (B95.8) or nontransforming (P3HR1) virus strains

Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell lines have been converted to EBV genome positivity by in vitro infection with the transforming EBV strain B95.8 and with the nontransforming mutant strain P3HR1, which has a deletion in the gene encoding the nuclear antigen EBNA2. These B95.8- and P3HR1-converted lines have been compared for their patterns of expression of EBV latent genes (i.e., those viral genes constitutively expressed in all EBV-transformed lines of normal B-cell origin) and for their recognition by EBV-specific cytotoxic T lymphocytes (CTLs), in an effort to identify which latent gene products provide target antigens for the T-cell response. B95.8-converted lines on several different EBV-negative BL-cell backgrounds all showed detectable expression of the nuclear antigens EBNA1, EBNA2, and EBNA3 and of the latent membrane protein (LMP); such converts were also clearly recognized by EBV-specific CTL preparations with restriction through selected human leukocyte antigen (HLA) class I antigens on the target cell surface. The corresponding P3HR1-converted lines (lacking an EBNA2 gene) expressed EBNA1 and EBNA3 but, surprisingly, showed no detectable LMP; furthermore, these converts were not recognized by EBV-specific CTLs. Such differences in T-cell recognition were not due to any differences in expression of the relevant HLA-restricting determinants between the two types of convert, as shown by binding of specific monoclonal antibodies and by the susceptibility of both B95.8 and P3HR1 converts to allospecific CTLs directed against these same HLA molecules. The results suggest that in the normal infectious cycle, EBNA2 may be required for subsequent expression of LMP and that both EBNA2 and LMP (but not EBNA1 or EBNA3) may provide target antigens for the EBV-specific T-cell response.     

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.     

Distinction between Epstein-Barr virus type A (EBNA 2A) and type B (EBNA 2B) isolates extends to the EBNA 3 family of nuclear proteins.

The Epstein-Barr virus (EBV) nuclear antigens EBNA 3a, 3b, and 3c have recently been mapped to adjacent reading frames in the BamHI L and E fragments of the B95.8 EBV genome. We studied by immunoblotting the expression of the family of EBNA 3 proteins in a panel of 20 EBV-transformed lymphoblastoid cell lines (LCLs) carrying either type A (EBNA 2A-encoding) or type B (EBNA 2B-encoding) virus isolates. Certain human sera from donors naturally infected with type A isolates detected the EBNA 3a, 3b, and 3c proteins in all type A virus-transformed LCLs (with a single exception in which EBNA 3b was not detected) but detected only EBNA 3a in LCLs carrying type B isolates. These results were confirmed with human and murine antibodies with specific reactivity against sequences of the type A EBNA 3a, 3b, or 3c expressed in bacterial fusion proteins. Conversely, selected human sera from donors naturally infected with type B strains of EBV identified the EBNA 3a encoded by both types of isolates plus two novel EBNAs present only in type B, and not in type A, virus-transformed LCLs; these novel proteins appear to be the type B homologs of EBNA 3b and 3c. The distinction between type A and type B EBV isolates therefore extends beyond the EBNA 2 gene to the EBNA 3 family of proteins. This has important implications with respect to the evolutionary origin of these two EBV types and also places in a new light recent studies which identified differences between type A and type B transformants in terms of growth phenotype (A. B. Rickinson, L. S. Young, and M. Rowe, J. Virol. 61:1310-1317, 1987) and of detection by EBV-specific cytotoxic T cells (D. J. Moss, I. S. Misko, S. R. Burrows, K. Burman, R. McCarthy, and T. B. Sculley, Nature [London] 331:719-721, 1988).     

Epstein-Barr virus-specific cytotoxic T-cell recognition of transfectants expressing the virus-coded latent membrane protein LMP

Cytotoxic T cells from Epstein-Barr virus (EBV)-immune individuals specifically kill EBV-transformed B cells from HLA class I antigen-matched donors even though the latently infected cells express only a restricted set of virus genes. The virus-induced target antigens recognized by these immune T cells have not been identified. In our experiments, EBV DNA sequences encoding the virus latent gene products Epstein-Barr nuclear antigen (EBNA)1, EBNA 2, and EBNA-LP and the latent membrane protein (LMP) were individually expressed in a virus-negative human B-lymphoma cell line, Louckes. Transfected clones expressing LMP were killed by EBV-specific cytotoxic T-cell preparations from each of three virus-immune donors HLA matched with Louckes through HLA-A2, B44 antigens; control transfectants or clones expressing one of the EBNA proteins were not recognized. Expression of LMP in a second virus-negative B-cell line, BL41, sensitized these cells to EBV-specific cytolysis restricted through the HLA-A11 antigen. To distinguish between the viral protein and an induced human B-cell activation antigen as the target for T-cell recognition, LMP was then expressed in a murine mastocytoma cell line, P815-A11-restricted human T cells. The LMP-expressing P815-A11 transfectants were susceptible to lysis by EBV-specific cytotoxic T cells from three HLA-A11-positive individuals. Both Louckes and P815-A11 cells were also transfected with constructs capable of encoding a truncated form of LMP (Tr-LMP) which lacks the N-terminal 128 amino acids of the full-length protein. Tr-LMP-expressing transfectants were not recognized by the above T-cell preparations. The results suggest that LMP, and, in particular, epitopes derived from the N-terminal region of the protein, provides one of the target antigens for the EBV-induced human cytotoxic T-cell response.     

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.     

Partial purification of the Epstein-Barr virus nuclear antigen(s)

The Epstein-Barr virus nuclear antigen (EBNA) is speculated to be involved in cell transformation by the virus. Studies on the molecular properties of EBNA, however, have yielded conflicting results. In this study, three Epstein-Barr virus(EBV)-induced antigens were isolated and purified from extracts prepared from Raji cells. These antigens were able to block the anticomplement immunofluorescence reaction, indicating that all three were related to EBNA. The soluble antigen was found wholly in the cytosol fraction. An EBV-induced nuclear antigen I was found both in the cytosol and the nucleus. The EBV-induced nuclear antigen II was found associated with the chromatin. The soluble antigen and the nuclear antigen I were separated and partially purified using phosphocellulose chromatography. Each was further purified 1,400-fold with respect to the whole cell state by chromatography on CL-Sepharose 6B followed by blue dextran-Sepharose. subunit molecular weights of 70,000 were determined for each of these antigens, both in the crude and purified state, by radioimmunoelectrophoresis and gel filtration. The nuclear antigen II was purified 2,500-fold using hydroxylapatite, CL-Sepharose 6B, and blue dextran-Sepharose chromatographies. This antigen displayed two subunits by radioimmunoelectrophoresis with molecular weights of 65,000 and 70,000. Although all antigens shared similar molecular weights, the extent of their homology remains to be determined.     

Identification and characterization of a cellular protein that cross-reacts with the Epstein-Barr virus nuclear antigen.

A 62,000-dalton (62K) cell protein reacts with antisera to the 72K polypeptide of the Epstein-Barr virus nuclear antigen (EBNA) in immunoblots. This protein was initially detected in EBNA-negative as well as EBNA-positive cell lines with anti-EBNA-positive human sera. A monoclonal antibody raised against the 72K EBNA and an antiserum from a rabbit immunized with the glycine-alanine domain of EBNA also reacted with the cellular protein. The cellular protein was partially purified from Epstein-Barr virus genome-positive and -negative cell lines. Absorption experiments identified a shared antigenic determinant between the 72K EBNA and 62K cellular protein. A comparison of the 62K protein and EBNA by protease digestion did not reveal similar peptides.     

Different Patterns of Epstein-Barr Virus Latency in Endemic Burkitt Lymphoma (BL) Lead to Distinct Variants within the BL-Associated Gene Expression Signature

Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as “molecular BL” (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.     

Antigens and DNA of a chimpanzee agent related to Epstein-Barr virus.

Biological and biochemical studies of the herpesvirus of chimpanzees previously demonstrated to be antigenically related to human Epstein-Barr virus (EBV) indicated that the agent is similar to EBV in that: (i) leukocyte culture of chimpanzees whose sera contained antibody against EBV capsid antigen could yield long-term lymphoblastoid cell lines (Ch-LCL) with B-cell characteristics; (ii) the DNA of Ch-LCL contained sequences homologous to approximately 35 to 45% of human EBV; (iii) Ch-LCL contained an intranuclear antigen, Ch-NA, that could be identified with some chimpanzee or orangutan serum in anticomplimentary immunofluorescence assays; and (iv) treatment of Ch-LCL with iododeoxyuridine resulted in expression of new antigenic activity that reacted with EA+ but not EA- human sera. Two lines of evidence indicate that the chimpanzee agent, although related to human EBV, is a distinct agent: (i) Ch-NA was antigenically distinct from EBV-rebv infection although it cross-reacts of a limited extent with a minor component of EBNA; and (ii) Ch-LCL are missing 55 to 65% of the DNA sequences of human EBV.     

Identification of the coding region for a second Epstein-Barr virus nuclear antigen (EBNA 2) by transfection of cloned DNA fragments.

Cell lines were established by co-transfection of cloned M-ABA Epstein-Barr virus (EBV) DNA fragments with plasmids conferring resistance to dominant selective markers. A baby hamster kidney cell line carrying the HindIII-I1 fragment exhibits a nuclear antigen of 82 000 daltons, serologically defined as EBV-determined nuclear antigen (EBNA) 1. Furthermore, a Rat-1 cell line transfected with DNA of the clone pM 780-28 containing three large internal repeats (BglII-U) and the adjacent BglII-C fragment expresses a nuclear antigen of 82 000 daltons which can be visualized only by a subset of anti EBNA-positive human sera. Sera recognizing the 82 000-dalton protein of the transfected cell line reacted with a protein of the same size in the non-producer line Raji, designated as EBNA 2. Conversely, sera without reactivity to the 82 000-dalton protein failed to react with EBNA 2 of Raji cells. P3HR-1 and Daudi cells with large deletions in BglII-U and -C are devoid of EBNA 2. The data presented provide evidence that a second EBNA protein is encoded by the region of the EBV genome which is deleted in the non-transforming P3HR-1 strain.     

Antibodies against synthetic peptides react with the second Epstein-Barr virus-associated nuclear antigen.

Five peptides were synthesized on the basis of amino acid sequences predicted from the transformation-associated BamHI WYH region of the genome of the Epstein-Barr virus (EBV). Antisera to two peptides deduced from a 1.6-kb open reading frame in the BamHI H fragment identified an 87 000-dalton nuclear polypeptide that was present in EBV-carrying cell lines that expressed the second EBV-determined nuclear antigen (EBNA-2). This polypeptide was not detected in cell lines that carried EBV variants with a deleted BamHI WYH region or in EBV-negative cell lines. Three peptides deduced from the 1.6-kb open reading frame reacted with human EBNA-positive sera, but not with EBNA-negative sera. Following affinity purification with the peptides, two of the corresponding human antibodies also reacted with the 87 000-dalton polypeptide.     

Host-cell-determined methylation of specific Epstein-Barr virus promoters regulates the choice between distinct viral latency programs.

Epstein-Barr virus (EBV) is capable of adopting three distinct forms of latency: the type III latency program, in which six EBV-encoded nuclear antigens (EBNAs) are expressed, and the type I and type II latency programs, in which only a single viral nuclear protein, EBNA1, is produced. Several groups have reported heavy CpG methylation of the EBV genome in Burkitt's lymphoma cell lines which maintain type I latency, and loss of viral genome methylation in tumor cell lines has been correlated with a switch to type III latency. Here, evidence that the type III latency program must be inactivated by methylation to allow EBV to enter the type I or type II restricted latency program is provided. The data demonstrates that the EBNA1 gene promoter, Qp, active in types I and II latency, is encompassed by a CpG island which is protected from methylation. CpG methylation inactivates the type III latency program and consequently allows the type I or II latency program to operate by alleviating EBNA1-mediated repression of Qp. Methylation of the type III latency EBNA gene promoter, Cp, appears to be essential to prevent type III latency, since EBNA1 is expressed in all latently infected cells and, as shown here, is the only viral antigen required for activation of Cp. EBV is thus a pathogen which subverts host-cell-determined methylation to regulate distinct genetic programs.     

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.     

Leukocyte migration inhibition studies with Epstein-Barr virus (EBV) determined nuclear antigen (EBNA) in relation to the EBV-carrier status of the donor

Cell extracts from EBV-genome-carrying cell lines inhibit the migration of leukocytes from EBV-positive but not seronegative healthy donors. In the present study extracts from EBV-negative lines and their own in vitro EBV-converted sublines were used to induce migration inhibition with leukocytes from seronegative and seropositive individuals. A clear difference was found between the extracts from EBV-negative and positive cell lines. Significant migration inhibition could be obtained with antigen(s) associated with the virus nonproducer state. Since EBNA is known to be expressed by all nonproducer EBV-genome-carrying cells, we have compared the effect of partially purified EBNA and correspondingly prepared mock-EBNA on the leukocyte migration. Purified EBNA inhibited the leukocyte migration of EBV seropositives, whereas mock-EBNA had no such effect.     

Differences in human cell lines to support stable replication of Epstein-Barr virus-based vectors

Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells.