SummaryEpstein-Barr virus (EBV), a human herpes virus with oncogenic potential, persists in B lymphoid tissues and is controlled by virus-specific cytotoxic T lymphocyte (CTL) surveillance. On reactivation in vitro, these CTLs recognize EBV-transformed lymphoblastoid cell lines (LCLs) in an HLA class I antigen-restricted fashion, but the viral antigens providing target epitopes for such recognition remain largely undefined. Here we have tested EBV-induced polyclonal CTL preparations from 16 virus-immune donors on appropriate fibroblast targets in which the eight EBV latent proteins normally found in LCLs (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, leader protein [LP], and latent membrane protein [LMP] 1 and 2) have been expressed individually from recombinant vaccinia virus vectors. Most donors gave multicomponent responses with two or more separate reactivities against different viral antigens. Although precise target antigen choice was clearly influenced by the donor's HLA class I type, a subset of latent proteins, namely EBNA 3A, 3B, and 3C, provided the dominant targets on a range of HLA backgrounds; thus, 15 of 16 donors gave CTL responses that contained reactivities to one or more proteins of this subset. Examples of responses to other latent proteins, namely LMP 2 and EBNA 2, were detected through specific HLA determinants, but we did not observe reactivities to EBNA 1, EBNA LP, or LMP 1. The bulk polyclonal CTL response in one donor, and components of that response in others, did not map to any of the known latent proteins, suggesting that other viral target antigens remain to be identified. This work has important implications for CTL control over EBu malignancies where virus gene expression is often limited to specific subsets of latent proteins. CTLs can play an important role in controlling virus infections, particularly as effectors of long-term immune surveillance against viruses that persist in the infected host. This is reflected in the frequency with which reactivation of persistent infections is observed in patients whose CTL responses are suppressed (1). Work in model systems first showed that the dominant components of virns-induced CTL populations are CD8 + MHC class I-restricted T cells (2) and that these effectors recognize peptide fragments of endogenously synthesized viral antigens presented on the target cell surface as a complex with MHC class I molecules (3, 4). In seeking to understand viral infections of humans, therefore, it is important in each case to know both the range of viral antigens that can induce effective CTL responses, and the influence of HLA class I polymorphism upon viral target antigen choice.The present study concerns human CTL responses to EBV. This lymphotropic herpes virus has potent cell growth-transforming activity both in vivo and in vitro, is the causative agent of infectious mononucleosis, and is strongly linked to at least three lymphoid malignancies: endemic Burkitt's lymphoma, the immunoblastic B cell lymphomas seen in immunocompromised patien...

SummaryTo examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-cx and -/3 chains from HLA-B8-restricted, CD8 + cytotoxic T cell clones specific for an immunodominant epitope (FLRGRAYGL) in Epstein-Barr virus (EBV) nuclear antigen 3. Herein, we show that identical TCR protein sequences are used by dones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of c~ and/3 chain rearrangements suggests that a persistent viral infection can select for a highly focused memory response and indicates a strong bias in gene segment usage and recombination. A novel double-step semiquantitative polymerase chain reaction (PCR) procedure and direct sequencing of amplified TCR cDNA from fresh lymphocytes derived from three HLA-B8 individuals detected transcripts specific for the conserved/3 chain in an EBV-seropositive donor but not in two seronegative donors. This report describes an unprecedented degree of conservation in TCR selected in response to a natural persistent infection.

Summal~There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type I and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA I as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the downregulation of latent antigens can be reversed.

Recombinant Epstein-Barr viruses (EBV) with a translation termination codon mutation inserted into the nuclear protein 3A (EBNA-3A) or 3C (EBNA-3C) open reading frame were generated by second-site homologous recombination. These mutant viruses were used to infect primary B lymphocytes to assess the requirement of EBNA-3A or-3C for growth transformation. The frequency of obtaining transformants infected with a wild-type EBNA-3A recombinant EBV was 10 to 15%. In contrast, the frequency of obtaining transformants infected with a mutant EBNA-3A recombinant EBV was only 1.4% (9 mutants in 627 transformants analyzed). Transformants infected with mutant EBNA-3A recombinant virus could be obtained only by coinfection with another transformation-defective EBV which provided wild-type EBNA-3A in trans. Cells infected with mutant EBNA-3A recombinant virus lost the EBNA-3A mutation with expansion of the culture. The decreased frequency of recovery of the EBNA-3A mutation, the requirement for transformation-defective EBV coinfection, and the inability to maintain the EBNA-3A mutation indicate that EBNA-3A is essential or critical for lymphocyte growth transformation and that the EBNA-3A mutation has a partial dominant negative effect. Five transformants infected with mutant EBNA-3C recombinant virus EBV were also identified and expanded. All five also required wild-type EBNA-3C in trans. Serial passage of the mutant recombinant virus into primary B lymphocytes resulted in transformants only when wild-type EBNA-3C was provided in trans by coinfection with a transformationdefective EBV carrying a wild-type EBNA-3C gene. A secondary recombinant virus in which the mutated EBNA-3C gene was replaced by wild-type EBNA-3C was able to transform B lymphocytes. Thus, EBNA-3C is also essential or critical for primary B-lymphocyte growth transformation. Epstein-Barr virus (EBV) is the etiological agent of infectious mononucleosis and is associated with several malignancies, including lymphomas in immunocompromised hosts (9, 70), Burkitt lymphoma (19, 35, 49, 71), nasopharyngeal carcinoma (14, 20, 71), and Hodgkin's disease (4, 62). EBV infects B lymphocytes and certain epithelial cells (27, 33, 59). EBV infection of B lymphocytes causes cell activation (7, 16, 38, 64, 68), continuous cell proliferation, and cell growth transformation as assayed in marmosets or SCID mice (43, 44, 55, 61, 67). Two EBV types, 1 and 2, coexist in most human populations. The two types have distinctive nuclear protein EBNA-LP,-2,-3A,-3B, and-3C genes (1, 13, 54, 56). Type 1 EBV is very efficient at transforming B lymphocytes, while type 2 EBV is very inefficient (52). This type-specific difference in transformation efficiency is primarily due to type-specific differences in the EBNA-2 gene (11, 52). Although EBV can potentially code for 80 to 100 genes (5), only a restricted set of 10 genes are expressed during B-lymphocyte latent infection (reviewed in references 29 and 30). Six genes encode EBNA-LP,-1,-2,-3A,-3B, and-3C. Two genes encode latent infection memb...

SUMMARYMonoclonal antibodies specific for the 'latent membrane protein' (LMP) of EpsteinBarr virus (EBV), one of the effector proteins of EBV-induced B cell transformation, have been generated from mice immunized with a fl-galactosidase fusion protein containing the carboxyl half of the B95.8 strain LMP sequence. Four monoclonal IgG1 antibodies, designated CS. 1, CS. 2, CS. 3 and CS. 4, which together recognized at least three different epitopes on the molecule, were used to examine various aspects of LMP expression in B cell lines transformed in vitro. The pooled CS. 1 to 4 reagent detected the LMPs encoded by each of 20 geographically distinct EBV isolates, despite a degree of inter-isolate heterogeneity in the size and antigenicity of the protein. In cell lines carrying the prototype B95.8 virus strain, particularly if these were virus producers, an additional lower molecular weight LMP was also detected; this appeared to correspond to the truncated form of the protein already predicted to exist from the analysis of B95.8 lytic cycle mRNAs. Attempts were made to identify an analogous truncated form of LMP in cell lines carrying other virus isolates after treatment with phorbol ester and/or sodium butyrate to induce virus production. Surprisingly these experiments showed that expression of the full length LMP molecule was itself strongly inducible by these agents; when monitored at the single cell level, this was a generalized response and was not restricted to cells entering a lytic cycle. Expression of LMP in EBVtransformed B cells therefore appears to be subject to a distinct type of regulation.

The entire Epstein-Barr virus genome is integrated into Burkitt tumor cell DNA at the terminal direct repeat sequence of the virus. There is no homology between the GC-rich (G, guanine; C, cytosine) terminal repeat and the AT-rich (A, adenine; T, thymine) cell sequences with which it has recombined. More than 15 kilobases of cell DNA have been deleted and 236 base pairs are duplicated at one virus-cell junction site.

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.

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.