Oxazolidinones mechanism of action: inhibition of the first peptide bond formation.

Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA templates but have no significant effect on poly(A)-dependent translation. Here we show that various oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70 S ribosomes using initiator-tRNA or N-protected CCA-Phe as a P-site substrate and puromycin as an A-site substrate. Steady-state kinetic analysis shows that oxazolidinones display a competitive inhibition pattern with respect to both the P-site and A-site substrates. This is consistent with a rapid equilibrium, ordered mechanism of the peptidyltransferase reaction, wherein binding of the A-site substrate can occur only after complex formation between peptidyltransferase and the P-site substrate. We propose that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiator fMet-tRNA(i)(Met) to the ribosomal peptidyltransferase P-site, which is vacant only prior to the formation of the first peptide bond.

A scintillation proximity assay for rna detection.

A homogeneous scintillation proximity assay (SPA) for detection of RNA transcripts is described. 3H-labeled RNA transcripts are hybridized in solution to biotinylated oligodeoxynucleotides (ODNs), which are then bound by streptavidin-coated, scintillant-embedded beads. Only bound 3H-labeled RNA transcripts are brought in close enough proximity to stimulate light emission from the beads. The results from this novel homogeneous assay correlated well with those obtained using the traditional filter-binding methods to measure RNA polymerase activity. The assay has been miniaturized to a 384-well format compatible with automated high-throughput screening. This SPA method has also been successfully used to probe RNA-accessible sites to hybridization, and thus should provide a useful tool for selecting effective antisense ODNs in antisense research.

Type 2 cytokines predominate in the human CD4(+) T-lymphocyte response to Epstein-Barr virus nuclear antigen 1.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persistently infects 85% of the adult population worldwide. In this report, we examine the proliferative response and cytokine secretion profile of CD4(+) T lymphocytes from a panel of unrelated EBV-positive donors against two EBV latent antigens, EBNA1 and EBNA3C. Substantial proliferative responses by CD4(+) lymphocytes were demonstrated to both antigens in multiple, randomly selected donors. Surprisingly, we observed a striking and consistent difference in cytokine response to EBNA1 and EBNA3C. EBNA1-specific CD4(+) T lymphocytes from multiple unrelated donors preferentially produced type 2-like cytokines in response to antigenic stimulation, while the response to EBNA3C was a characteristic type 1 response. The implications of these findings for EBV persistence and the development of EBV-associated malignancies are discussed.

Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1.

The Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1 is critical for the persistence of the viral episome in replicating EBV-transformed human B cells. Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA1 is invisible to CD8(+) cytotoxic T lymphocytes because its Gly/Ala repeat domain prevents proteasome-dependent processing for presentation on major histocompatibility complex (MHC) class I. We now describe that CD4(+) T cells from healthy adults are primed to EBNA1. In fact, among latent EBV antigens that stimulate CD4(+) T cells, EBNA1 is preferentially recognized. We present evidence that the CD4(+) response may provide a protective role, including interferon gamma secretion and direct cytolysis after encounter of transformed B lymphocyte cell lines (B-LCLs). Dendritic cells (DCs) process EBNA1 from purified protein and from MHC class II-mismatched, EBNA1-expressing cells including B-LCLs. In contrast, B-LCLs and Burkitt's lymphoma lines likely present EBNA1 after endogenous processing, as their capacity to cross-present from exogenous sources is weak or undetectable. By limiting dilution, there is a tight correlation between the capacity of CD4(+) T cell lines to recognize autologous B-LCL-expressing EBNA1 and DCs that have captured EBNA1. Therefore, CD4(+) T cells can respond to the EBNA1 protein that is crucial for EBV persistence. We suggest that this immune response is initiated in vivo by DCs that present EBV-infected B cells, and that EBNA1-specific CD4(+) T cell immunity be enhanced to prevent and treat EBV-associated malignancies.

High frequency of cytomegalovirus-specific cytotoxic T-effector cells in HLA-A*0201-positive subjects during multiple viral coinfections.

How the cellular immune response copes with diverse antigenic competition is poorly understood. Responses of virus-specific cytotoxic T lymphocytes (CTL) were examined longitudinally in an individual coinfected with human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CTL responses to all 3 viruses were quantified by limiting dilution analysis and staining with HLA-A*0201 tetrameric complexes folded with HIV-1, EBV, and CMV peptides. A predominance of CMV-pp65-specific CTL was found, with a much lower frequency of CTL to HIV-1 Gag and Pol and to EBV-BMLF1 and LMP2. The high frequency of CMV-specific CTL, compared with HIV-1- and EBV-specific CTL, was confirmed in an additional 16 HLA-A*0201-positive virus-coinfected subjects. Therefore, the human immune system can mount CTL responses to multiple viral antigens simultaneously, albeit with different strengths.

Presentation of epstein-barr virus latency antigens to CD8(+), interferon-gamma-secreting, T lymphocytes.

Epstein-Barr virus (EBV) infects more than 95 % of the human population and causes an asymptomatic life-long infection in the majority of EBV carriers. Cell-mediated immunity provides resistance to EBV, as demonstrated by the occurrence of EBV-induced post-transplant lymphoproliferative disease in immunosuppressed patients. Here we looked for IFN-gamma-producing T lymphocytes in the blood of healthy donors with a rapid enzyme-linked immunospot (ELISPOT) assay, comparing as antigen presenting cells monocytes and dendritic cells (DC) infected with recombinant vaccinia virus (rVV). We found a strong CD8(+) ELISPOT response to one or more of the EBNA 3A, 3B and 3C antigens in the PBMC from 14 / 18 donors. The sensitivity of the overnight ELISPOT assay was increased using DC as antigen-presenting cells, including 3 / 3 individuals who lacked ELISPOT in PBMC. In addition, DC could markedly expand EBV-specific spots after a 7-day culture. In a smaller number of donors, we documented recognition of the subdominant LMP 1, LMP 2 and EBNA 1 antigens that are expressed in a variety of EBV-associated malignancies. Therefore our data provide more evidence for the efficacy of DC in eliciting rapid responses to EBV latency antigens in circulating CD8(+) T cells.

Induction of Epstein-Barr virus-specific cytotoxic T-lymphocyte responses using dendritic cells pulsed with EBNA-3A peptides or UV-inactivated, recombinant EBNA-3A vaccinia virus.

Cell-mediated immunity, especially the cytotoxic T lymphocyte (CTL), provides resistance to Epstein-Barr virus (EBV), as is demonstrated by the occurrence of posttransplant lymphoproliferative disease in immunosuppressed patients. We set out to use dendritic cells (DCs) to elicit anti-EBV-specific CTLs in culture. In unselected, HLA-B8(+) donors, monocyte-derived mature DCs were pulsed with the HLA-B8-restricted EBNA-3A peptide, FLRGRAYGL, and added to autologous T cells for 7 days at a DC:T ratio of 1:5 to 1:60. The cultured cells specifically lysed EBNA-3A peptide-pulsed, HLA-B8(+), B-lymphoblastoid cell lines in a 5-hour (51)Cr-release assay. The generation of CTLs did not require the addition of interleukin-2. In comparison, monocytes were weak antigen-presenting cells. DCs were then infected with recombinant vaccinia-EBNA-3A. Vaccinia infection significantly decreased the viability of immature DCs after 3 days of culture (to 25% to 45%) but had a smaller effect on mature DC recovery (40% to 70%). To decrease these cytopathic effects and to expand the potential use of vaccinia vectors for DC therapy in immunocompromised patients, we successfully used psoralen and UV-inactivated virus. Mature DCs pulsed with either live or inactivated vaccinia EBNA-3A virus could elicit strong EBNA-3A-specific CTLs. Therefore, mature DCs are powerful stimulators of EBV-specific CTLs and their major histocompatibility complex class I products can even be charged with UV-inactivated recombinant vaccinia.

Localization of the binding site for transforming growth factor-beta in human alpha2-macroglobulin to a 20-kDa peptide that also contains the bait region.

alpha2-Macroglobulin (alpha2M) functions as a major carrier of transforming growth factor-beta (TGF-beta) in vivo. The goal of this investigation was to characterize the TGF-beta-binding site in alpha2M. Human alpha2M, which was reduced and denatured to generate 180-kDa subunits, bound TGF-beta1, TGF-beta2, and NGF-beta in ligand blotting experiments. Cytokine binding was not detected with bovine serum albumin that had been reduced and alkylated, and only minimal binding was detected with purified murinoglobulin. To localize the TGF-beta-binding site in alpha2M, five cDNA fragments, collectively encoding amino acids 122-1302, were expressed as glutathione S-transferase (GST) fusion proteins. In ligand blotting experiments, TGF-beta2 bound only to the fusion protein (FP3) that includes amino acids 614-797. FP3 bound 125I-TGF-beta1 and 125I-TGF-beta2 in solution, preventing the binding of these growth factors to immobilized alpha2M-methylamine (alpha2M-MA). The IC50 values were 33 +/- 5 and 26 +/- 6 nM for TGF-beta1 and TGF-beta2, respectively; these values were comparable with or lower than those determined with native alpha2M or alpha2M-MA. A GST fusion protein that includes amino acids 798-1082 of alpha2M (FP4) and purified GST did not inhibit the binding of TGF-beta to immobilized alpha2M-MA. FP3 (0.2 microM) neutralized the activity of TGF-beta1 and TGF-beta2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3 also increased NO synthesis by RAW 264.7 cells, mimicking an alpha2M activity that has been attributed to the neutralization of endogenously synthesized TGF-beta. Thus, we have isolated a peptide corresponding to 13% of the alpha2M sequence that binds TGF-beta and neutralizes the activity of TGF-beta in two separate biological assays.

Targeting Epstein-Barr virus nuclear antigen 1 (EBNA1) through the class II pathway restores immune recognition by EBNA1-specific cytotoxic T lymphocytes: evidence for HLA-DM-independent processing.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all malignancies associated with EBV and there is now convincing evidence to suggest that EBNA1 is not recognized by MHC class I-restricted cytotoxic T lymphocytes (CTL). The lack of recognition of EBNA1 has been attributed to a cis-acting inhibitory effect of glycine-alanine repetitive (G-Ar) sequences on the endogenous processing of this antigen through the class I pathway. In the present study we have explored the possibility of targeting EBNA1 through an alternative mechanism using the MHC class II pathway. Using purified EBNA1 protein, we demonstrate here that CD4+ CTL can efficiently recognize EBV-transformed B cells and Burkitt's lymphoma cells following exogenous sensitization with this antigen, and this immune recognition is not affected by the G-Ar domain within EBNA1. Analysis of the processing mechanism revealed that intracellular loading of class II molecules with an EBNA1 epitope occurs through an HLA-DM-independent pathway. These results highlight a novel mechanism for immune recognition of EBNA1 and also demonstrate that the G-Ar-mediated protection from processing can be overridden if this antigen is presented through the class II pathway.

Cytotoxic CD8+ T cells recognize EBV antigen but poorly kill autologous EBV-infected B lymphoblasts: immunodominance is elicited by a peptide epitope that is presented at low levels in vitro.

CD8+ T cells play a crucial role in surveillance against EBV-associated malignancies. EBV-specific T cells traditionally have been identified by their ability to kill autologous EBV-transformed B lymphoblastoid cell lines (LCL). Here we report CD8+ cloned and bulk T cells that specifically recognize EBV nuclear Ag EBNA-3C, but do not efficiently kill autologous or HLA-matched LCL. The low cytolysis of these T cells was due to the extremely low density of the antigenic epitope (LDFVRFMGV, EBNA-3C amino acids 285-293) on autologous LCL. The T cells efficiently killed target cells in the presence of < 1 pM synthetic EBNA-3C peptide and, therefore, recognize peptide/HLA complexes with high avidity. Donor T cells with this phenotype were stimulated by autologous LCL and dominated the in vitro EBV-specific response. This indicates that low abundance viral peptides can induce a dominant T cell response.

Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response.

Epstein-Barr virus (EBV), a human gamma-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8+ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8+ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBV-replicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in alpha- and beta-herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of gamma-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.

Transient selection during vaccinia virus recombination with insertion vectors without selectable markers.

Isolation of recombinant viruses after DNA transfection remains the most time-consuming and labor-intensive feature in the production of vaccinia virus (VV) recombinants. This procedure has been aided by the design and incorporation of both positive and negative selectable markers into the insertion vectors. Use of these selectable markers is not always feasible or desirable. In the absence of selectable markers, individual plaque characterization is required. A method of transient selection during transfection and recombination is described that does not require that any selectable marker be present in the insertion vector sequence. This method relies on the co-transfection of a guanine phosphoribosyl transferase (gpt) selectable marker on an unlinked plasmid into VV-infected cells pretreated for gpt selection. Following recombination, the output virus can be plated on cells without any further selection and screened for recombinant virus. While the total recombinant virus population is slightly reduced, the total progeny virus is reduced even further, resulting in more than sevenfold enhancement in the frequency of recombinant virus, which allows for more efficient identification.

Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing.

Epstein-Barr virus (EBV)-induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.

Natural variants of the immunodominant HLA A11-restricted CTL epitope of the EBV nuclear antigen-4 are nonimmunogenic due to intracellular dissociation from MHC class I:peptide complexes.

EBV isolates from human populations with a high frequency of HLA A11 evade recognition by CTLs specific for an immunodominant A11-restricted epitope derived from the EBV nuclear antigen 4 (EBNA-4). We have previously described four nonimmunogenic variants of this epitope carrying single amino acid substitutions in the anchor residues of the peptide. We have now investigated the antigenicity, A11 binding capacity, endoplasmic reticulum translocation, endogenous processing, and presentation of these variants. The nonimmunogenic peptides were either unable to bind to HLA A11 or formed complexes of significantly lower stability compared with the immunogenic epitope. The latter peptides were produced in relatively large amounts by endogenous processing of EBNA-4 and associated with A11 molecules almost as efficiently as the immunogenic epitope, but the complexes failed to accumulate at the cell surface. The defect was not reversed by incubation of lymphoblastoid cell lines carrying the variant EBV strains at 26 degrees C. CTL lysis of HLA A11 positive targets was achieved by expressing one of the nonimmunogenic peptides through a vaccinia recombinant. However, the amount of peptide required for CTL sensitization exceeded, by at least 30-fold, that required for recognition of the immunogenic epitope. Collectively, these results suggest that complexes containing the nonimmunogenic peptides are formed but are then destroyed intracellularly. Thus, a specialized sorting mechanism seems to contribute in shaping the repertoire of peptides presented to T lymphocytes.

Defective presentation of MHC class I-restricted cytotoxic T-cell epitopes in Burkitt's lymphoma cells.

Defects of antigen processing/presentation have been suggested to play a role in the escape of Burkitt's lymphoma (BL) from cytotoxic T lymphocyte (CTL)-mediated rejection. Impaired presentation of an immunodominant HLA A11-restricted epitope from the resident or recombinant vaccinia virus-expressed Epstein-Barr virus nuclear antigen (EBNA)4 was demonstrated in the EBV-positive E95B-BL28 and its EBV-negative parental BL28 cell lines. We have investigated whether this was due to (i) impaired production of the antigenic peptide, (ii) poor peptide translocation into the ER lumen or (iii) inefficient maturation and transport of the MHC-peptide complexes at the cell surface. The defect was not overcome by cytosolic expression of a pre-formed epitope, suggesting that presentation of EBNA4 is not limited by inefficient production of the antigenic peptide. BL28 expressed 5- to 10-fold lower levels of the transporter associated with antigen presentation (TAP) 1 and TAP2 proteins and behaved poorly in a streptolysin-O-mediated peptide translocation assay, whereas E95B-BL28 showed higher TAP expression and virtually normal transporter function. Up-regulation of HLA A11 and reconstitution of TAP activity by treatment with IFN-gamma did not restore presentation of the resident EBNA4 in E95SB-BL28 and did not enhance presentation of the vaccinia virus-expressed intact protein or preformed epitope. Efficient maturation of class I molecules to Endo H-resistant species was demonstrated in pulse-chase experiments. Taken together, our findings identify a previously uncharacterized defect of antigen presentation which appears to affect events occurring after proteasomal degradation but before TAP-dependent peptide transport and MHC class I assembly and maturation.

Recruitment during infectious mononucleosis of CD3+CD4+CD8+ virus-specific cytotoxic T cells which recognise Epstein-Barr virus lytic antigen BHRF1.

Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis (IM) which is a common sequel to primary EBV infection. Thereafter, the virus is maintained as a lifetime latent infection. Although the proteins expressed during the latent EBV infection provide a rich source of immunogenic epitopes, very little is known about cytotoxic T lymphocyte (CTL) control of primary EBV infection. The present report is based on an analysis of CTL clones derived from a patient suffering from acute IM. An intriguing feature of six CTL clones that displayed an HLA-restricted pattern of cell lysis was their initial coexpression of the T cell markers CD3, CD4, and CD8. Detailed analysis of one of these clones, which was restricted through the class II MHC antigen DR2, revealed reactivity with an epitope within the EBV lytic cycle early antigen, BHRF-1, which corresponds to the C-terminal region of the protein (AGLTLSLLVICSYLFISRG) (residues 171-189). There have been no previously published reports describing a CTL response during acute IM directed against an EBV lytic antigen. Interestingly, the coexpression of CD4 and CD8 by these CTLs during acute IM suggests that CD3+CD4+CD8+ cortical thymocytic precursor cells are recruited in order to overcome the EBV infection.

Isolation of cytotoxic T lymphocytes from healthy seropositive individuals specific for peptide epitopes from Epstein-Barr virus nuclear antigen 1: implications for viral persistence and tumor surveillance.

The question of whether Epstein-Barr nuclear antigen 1 (EBNA1) includes cytotoxic T lymphocyte (CTL) epitopes has generated considerable scientific interest, primarily due to its important implications for the overall biology of Epstein-Barr virus (EBV). Earlier studies have suggested that EBV-associated malignancies that express only EBNA1 escape virus-specific immune surveillance since this antigen is not a target for CTL recognition. In the present report we have used a modified protocol to demonstrate that EBNA1 includes sequences which can be recognized by both polyclonal and clonal CTLs. CD4+ CTL clones were isolated from a healthy, seropositive donor that recognized the peptide epitope TSLYNLRRGTALA from EBNA1 in association with HLA DR1. Interestingly, these CTLs are unable to lyse EBV-infected B cells suggesting that EBNA1 may not be endogenously processed and/or presented to the host CTL response. Despite recent suggestions that glycine-alanine repeat sequences within EBNA1 can inhibit endogenous processing, target cells infected with recombinant vaccinia vectors encoding truncated EBNA1 proteins, without these repeat sequences, were not recognized by this CTL clone. Thus it seems that the presence of glycine-alanine repeats is not responsible for inhibiting the processing of the class II-restricted epitope defined in this study. These results substantiate the view that EBV-infected normal and malignant cells, where viral gene expression is limited to EBNA1, can resist CTL-mediated immune surveillance in vivo.

Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1.

The Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA1) is expressed in latently EBV-infected B lymphocytes that persist for life in healthy virus carriers, and is the only viral protein regularly detected in all malignancies associated with EBV. Major histocompatibility complex (MHC) class I-restricted, EBNA1-specific cytotoxic T lymphocyte (CTL) responses have not been demonstrated. Using recombinant vaccinia viruses encoding chimaeric proteins containing an immunodominant human leukocyte antigen A11-restricted CTL epitope, amino acids 416-424 of the EBNA4 protein, inserted within the intact EBNA1, or within an EBNA1 deletion mutant devoid of the internal Gly-Ala repetitive sequence, we demonstrate that the Gly-Ala repeats generate a cis-acting inhibitory signal that interferes with antigen processing and MHC class I-restricted presentation. Insertion of the Gly-Ala repeats downstream of the 416-424 epitope inhibited CTL recognition of a chimaeric EBNA4 protein. The results highlight a previously unknown mechanism of viral escape from CTL surveillance, and support the view that the resistance of cells expressing EBNA1 to rejection mediated by CTL is a critical requirement for EBV persistence and pathogenesis.

A novel Epstein-Barr virus glycoprotein gp150 expressed from the BDLF3 open reading frame.

Analysis of cDNAs mapping to the BamHI D fragment of the Epstein-Barr virus (EBV) genome indicates that the BDLF3 open reading frame, which is predicted to encode a type 1 membrane protein of 234 amino acids, is expressed as an unspliced message. Expression of the open reading frame as a recombinant protein in vaccinia virus reveals a glycoprotein that has both N- and O-linked sugars. Antibodies made to the recombinant protein immunoprecipitate a late glycoprotein with a mobility of approximately 150,000 Da from EBV-producing cells. The glycoprotein is associated with the virion. Antibodies to it appear to react primarily with carbohydrate and do not demonstrate neutralizing activity.

Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus.

To examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-alpha and -beta 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 clones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of alpha and beta chain rearrangements suggest 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 beta 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.