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M G Masucci

Publications and source records attributed to M G Masucci.

At least 73 records · Page 4Linked to original sources
PubMed1992

Patterns of Epstein-Barr virus infection in non-neoplastic lymphoid tissue.

Taking advantage of the abundant expression of the small Epstein-Barr virus (EBV)-encoded RNAs (EBERs) in latently infected cells, we have analyzed 72 normal and hyperplastic lymph nodes and three tonsils of acute infectious mononucleosis (IM) for the presence and distribution of EBV+ cells using EBER-specific in situ hybridization, in some cases combined with immunohistologic demonstration of cell type-characteristic antigens. In IM, large numbers of EBV+ lymphoid B blasts were detectable in extrafollicular areas, whereas germinal centers were generally free of EBV+ cells. In reactive lymph nodes, the frequency of EBV+ cells varied with the degree of lymphoid hyperplasia and underlying immune status. The lowest numbers of EBV+ cells were detected in nonactivated lymph nodes and highest in human immunodeficiency virus-associated lymphadenopathy. If present in these lymph nodes, EBV+ cells were almost exclusively localized to extrafollicular areas, as also observed in IM. However, in contrast to IM, these cells were mainly small lymphocytes. Furthermore, in some instances, occasional scattered EBV+ cells were seen within germinal centers, and in two cases diffuse expansions of EBV+ cells occurred within a single germinal center each, indicating that under certain circumstances EBV+ B lymphocytes may participate in physiologic germinal center reactions. These findings reflect the interference of EBV with physiologic lymphoid differentiation pathways and provide a link to EBV-associated malignant lymphomas with a postulated origin from germinal center cells.

B-Lymphocytes↗
PubMed1992

Immune escape by Epstein-Barr virus (EBV) carrying Burkitt's lymphoma: in vitro reconstitution of sensitivity to EBV-specific cytotoxic T cells.

Epstein-Barr virus (EBV) positive Burkitt's lymphoma (BL) cells are markedly less sensitive to EBV-specific cytotoxic T cell (CTL) recognition than EBV-transformed lymphoblastoid cell lines of normal B cell origin. Three features of the BL cell phenotype might contribute to this reduced susceptibility: (i) low expression of cell adhesion molecules, (ii) low expression of HLA class I and selective down-regulation of particular alleles, and (iii) down-regulation of all transformation-associated EBV antigens except EBV-encoded nuclear antigen (EBNA)-1. This study assesses the individual importance of each of these features for immune escape. For this purpose the WW1-BL cell line was used which expresses all the known transformation-associated EBV antigens (EBNA-1 to -6 and latent membrane protein-1 and -2) but which is negative for HLA A11 and for the adhesion molecule leukocyte function associated antigen-3 (LFA-3). Using recombinant vectors, these deficiencies have been sequentially corrected and the cells have been tested for sensitivity to EBV (B95.8 strain)-induced CTL preparations recognizing epitope(s) of EBNA-4 in the context of HLA A11. Expression of HLA A11 alone or in combination with LFA-3 did not sensitize WW1-BL cells to these effectors. Lysis was only achieved when HLA A11 was co-expressed with the B95.8 virus-encoded EBNA-4 protein, and in these circumstances sensitization did not require LFA-3. These results indicate that reconstitution of the relevant HLA-EBV epitope target complex on the cell membrane is sufficient to render BL cells sensitive to virus-specific cytolysis. The requirement for EBNA-4 reconstitution to achieve lysis of the WW1-BL/A11 transfectant suggested that the resident WW1 virus-encoded EBNA-4 protein did not contain the relevant target epitope for HLA A11-restricted recognition. This was confirmed by transferring the WW1 virus isolate into another A11-positive B cell background.

Antigens, CD↗
PubMed1992

T cell recognition of Epstein-Barr virus associated lymphomas.

Epstein-Barr virus, a lymphotropic herpesvirus of humans, has potent B cell growth transforming activity yet persists in the lymphoid tissues of most individuals as a lifelong asymptomatic infection. Virus induced B cell growth transformation in vitro is associated with the expression of a limited set of viral genes encoding six nuclear antigens (EBNA 1, 2, 3A, 3B, 3C and LP) and two latent membrane proteins (LMP 1, 2). Healthy virus carriers possess strong EBV specific CTL memory that can be reactivated in vitro. Here, we summarize experiments in which the antigenic specificities of these HLA class I restricted memory CTL responses have been mapped in a range of individuals with different HLA backgrounds. Of the known EBV latent proteins, EBNA 3A, 3B and 3C are frequently the dominant targets for such responses, but examples of responses directed against epitopes of EBNA 2, EBNA-LP or the LMP have been identified; by contrast, CTL responses against epitopes of EBNA 1 have not been observed. Epstein-Barr virus is associated with at least three malignancies of lymphoid origin--immunoblastic lymphomas of the immunosuppressed, endemic Burkitt's lymphoma and a subset of Hodgkin's disease. The immunoblastic lymphomas express the complete spectrum of EBV coded latent proteins and a cellular phenotype similar to that of in vitro transformed B lymphoblastoid cell lines; accordingly, they remain sensitive to EBV specific CTL recognition. Endemic BL cells are not recognized by such CTL, and at least three consistent features of this tumour could contribute to immune escape: (a) allele specific downregulation of HLA class I antigen expression, (b) absence/low expression of cellular adhesion molecules and (c) restriction of EBV latent protein expression to EBNA 1 only. The relative importance of these three features of the BL cell phenotype with regard to sensitivity to CTL recognition is re-interpreted in the light of recent results. Finally, the pattern of virus latent protein expression in EBV positive Hodgkin's disease is described, and the possibility of EBV specific CTL control against this tumour is discussed.

Antigens, Viral↗
PubMed1991

Stimulation with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines generates HLA class I-specific CTLs with different target cell avidity.

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) are potent inducers of cytotoxic T-lymphocytes (CTL) in allogeneic mixed lymphocyte cultures (MLC). The contribution of EBV antigens to the induction of cytotoxic responses was investigated by comparing CTL clones derived from allogeneic MLCs of lymphocytes from one EBV seropositive and one seronegative donor for their capacity to lyse paired EBV positive and negative targets. The majority of the clones showed a conventional "HLA-specific" cytotoxicity and lysed equally well HLA-matched LCLs and mitogen-induced T- or B-blasts. A minority of the clones from both donors exhibited an "LCL-selective" killing potential as they lysed poorly T- and B-blasts. The LCL-selective clones did not recognize EBV antigens because they could not discriminate between EBV negative Burkitt lymphoma (BL) lines and their in vitro EBV-converted sublines. MAbs to CD3, CD8, and MHC class I antigens blocked the lysis of LCLs by HLA-specific and LCL-selective CTLs with comparable efficiency suggesting that the two effector types express T-cell receptors of similar affinity. T-blasts were unable to inhibit the lysis of LCLs in cross competition assays. This correlated with a significantly lower expression of the cell adhesion molecules ICAM-1 and LFA-3. The results suggest that stimulation with allogeneic LCLs activates HLA class I-specific CTLs with variable target cell avidity. Only CTLs that act independently of the enhancing effect of cell adhesion molecules are able to lyse mitogen-induced T- and B-blasts.

Cell Adhesion Molecules↗
PubMed1991

The epstein-Barr-virus-encoded membrane protein LMP but not the nuclear antigen EBNA-1 induces rejection of transfected murine mammary carcinoma cells.

The EBV-encoded membrane protein (LMP) and the nuclear antigen EBNA-1 were compared for their capacity to induce rejection of transfected murine mammary carcinoma cells in syngeneic hosts. The tumorigenic potential of stable LMP and EBNA-1 expressing sublines of the ACA (H-2f)-derived mammary carcinoma line S6C was tested in pre-immunized syngeneic and semi-syngeneic animals. LMP expressing S6C cells elicited a strong rejection response as demonstrated by the lower tumor take and slower growth in immunized vs. control mice. In contrast, EBNA-1-expressing cells were non-immunogenic in syngeneic hosts and in one semi-syngeneic F1-hybrid. Rejection in 2 additional F1-hybrids did not appear to be due to EBNA-1-specific immune responses. Our findings support the hypothesis that the escape of EBV carrying tumor cells from EBV-specific immune surveillance may be facilitated by the fact that viral gene expression is limited to EBNA-1.

Animals↗
PubMed1991

Aberrant expression of HLA class-I antigens in Burkitt lymphoma cells.

HLA class-I expression has been investigated by biochemical methods in 14 Burkitt lymphoma (BL) cell lines and the corresponding Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the same individuals. Selective down-regulation of one or more HLA class-I specificities was demonstrated in 9 out of 14 BL lines. The defect was restricted to a single HLA-A allele in 3 of the lines (BL29, BL72, WW-I-BL). Four lines (BL28, BL37, BL41 and Jijoye M13) showed down-regulation of both HLA-A and -C alleles, and one (BL36) failed to express one HLA-C allele. Only one BL line (WW-2-BL) had lost one HLA-A and one HLA-B allele. The allele-specific defects were mainly detected in cell lines that had maintained the phenotypic characteristics of the original tumor. Expression of B-cell activation markers and the EBV-encoded nuclear antigen (EBNA)-2 correlated with up-regulation of the Cw4 allele in the P79 subline of the BL line Jijoye. Treatment with gamma-interferon (IFN) resulted in full or partial reversion of the HLA class-I defects in some of the cases but had no significant effect in others. This was not due to a cell-line-related unresponsiveness to IFN, nor did it reflect an allele-specific mode of regulation because the same allele could respond differently in different cell lines. The data suggest that defective expression of HLA class-I antigens, which appears to be more prevalent for alleles within the HLA-A and -C loci, is a common feature of BL cell lines. Different regulatory mechanisms appear to be involved.

Alleles↗
PubMed1991

Search for the critical characteristics of phenotypically different B cell lines, Burkitt lymphoma cells and lymphoblastoid cell lines, which determine differences in their functional interaction with allogeneic lymphocytes.

Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines (LCL). We studied three groups of BL lines for their capacity to interact with allogeneic lymphocytes. The results showed that as long as the lines have the group I phenotype, they do not stimulate allogeneic T lymphocytes irrespective whether they carry the EBV genome. The group II and III cells are stimulatory. Generally there was no correlation between sensitivity ot lymphocyte-mediated lysis and the phenotype of the lines. In one set of lines, the group I cells had higher sensitivity to both natural killer and lymphokine-activated killer effectors compared to the group II or III lines. However, such correlation could not be seen with the other two sets of lines. Among the phenotypic features investigated, expression of the adhesion molecules LFA-1 and LFA-3 correlated with the tendency for cell aggregation.

Antigens, Surface↗
PubMed1991

B cell phenotype-dependent expression of the Epstein-Barr virus nuclear antigens EBNA-2 to EBNA-6: studies with somatic cell hybrids.

The expression of the transformation-associated Epstein-Barr virus (EBV)-encoded nuclear antigens (EBNAs) 1 to 6 and membrane protein LMP-1 was studied in a series of somatic cell hybrids derived from the fusion of the EBV-transformed lymphoblastoid cell line (LCL) KR-4, and the EBV-carrying Burkitt's lymphoma lines Daudi, P3HR-1 and Raji, with non-B cell lines of fibroblast, erythroid, myeloid and epithelial origin. Expression of EBNAs 2 to 6 was down-regulated in the hybrids in parallel with extinction of the B cell markers CD19, CD20, CD21, CD23, HLA class II, and surface or cytoplasmic immunoglobulin. LMP-1 was expressed independently of EBNA-2 in hybrids derived by the fusion of the LMP-1-positive KR-4 and P3HR-1 cell lines with epithelial and myeloid cells, respectively. LMP-1 was down-regulated in hybrids derived by the fusion of P3HR-1 with an erythroid cell line and in the hybrid between Raji and a mouse fibrosarcoma line. EBNA-1 was the only EBV antigen that was regularly expressed in the hybrids regardless of the dominating cellular phenotype. The autonomous expression of EBNA-1 suggests that its regulatory pathway is independent of phenotype-associated cellular or viral factors. In contrast, the expression of EBNAs 2 to 6 appears to require a B cell environment. EBNA-2 was shown to contribute to the regulation of LMP expression in B cells. We show that in LCL-carcinoma hybrids the dominating epithelial phenotype is permissive for LMP expression in the absence of EBNA-2.

Antigens, Viral↗
PubMed1991

Cell phenotype dependent expression of MHC class I antigens in Burkitt's lymphoma cell lines.

Burkitt's lymphoma (BL) is a highly malignant B cell tumor characterized by three types of chromosomal translocation which constitutively activate the c-myc oncogene by juxtaposing it to Ig coding sequences. Epstein-Barr virus (EBV) infection, hyperendemic malaria and HIV-caused immunosuppression are thought to contribute to the pathogenesis of the tumor. Cell lines derived from EBV carrying and EBV negative BLs often show altered MHC class I antigen expression. The defects include a lower expression of all HLA class I antigens compared to EBV transformed normal B-blasts, and selective down-regulation of certain HLA-A and HLA-C alleles. As a consequence BL cells are often resistant to cytotoxic T lymphocyte (CTL) mediated destruction. Alleles selective down-regulations are found only in cell lines that maintain the tumor cell phenotype while shift towards a more activated 'B-blast like' phenotype is accompanied by HLA class I up-regulation. A similar pattern of HLA class I expression can be found in a subpopulation of germinal center B cells which express a 'BL like' phenotype. Our findings suggest that the HLA class I expression of BL cells reflects the characteristics of the normal B cell precursor and is probably not the result of immune selection.

Antigens, CD↗
PubMed1990

Expression of the Epstein-Barr virus (EBV)-encoded membrane antigen (LMP) increases the stimulatory capacity of EBV-negative B lymphoma lines in allogeneic mixed lymphocyte cultures.

Epstein-Barr virus (EBV)-negative Burkitt lymphoma (BL) lines are poor stimulators in allogeneic mixed lymphocyte cultures compared to EBV-transformed lymphoblastoid cell lines derived from the same individuals. We have previously shown that the stimulatory capacity of the tumor cells is increased after EBV conversion (Avila-Carino et al., Int. J. Cancer 1987. 40: 691). As a first step towards the identification of the viral gene product responsible for this change we have studied the influence of the EBV latent membrane protein (LMP) on the stimulatory capacity of the EBV-negative BL lines BL41 and DG75 and the B lymphoma line BJAB. Four LMP-transfected sublines of BL41, four DG75 LMP transfectants and one LMP-transfected subline of BJAB showed a significantly stronger stimulatory capacity than the original line. The effect was directly proportional to the amount of LMP detected in each transfectant but was not due to reactivation of LMP-specific memory cells since lymphocytes from EBV-seropositive and -seronegative individuals responded equally. In order to define the relation between LMP expression and induction of stimulatory capacity, DG75 was transfected with constructs containing the LMP gene under the control of an heat-shock promoter. The peak of LMP expression in heat shock-treated cells preceded the appearance of stimulatory capacity by 6-12 h suggesting that critical amounts of the protein may be required to induce the phenotypic change recognized by the T cells. LMP influenced in a dose-dependent manner the expression of the adhesion molecules LFA-1, LFA-3 and ICAM-1 and B cell activation markers CD23 and CD39 in transfected sublines of BL41, but did not affect the expression of these markers in the DG75 and BJAB cell line. All LMP-expressing transfectants showed an increased capacity to form conjugates with unprimed allogeneic lymphocytes.

Antibodies, Monoclonal↗
PubMed1990

High-resolution one-dimensional isoelectric focusing of mouse MHC class I antigens. Identification of natural and experimentally induced class I antigens.

A method is described for a biochemical comparison of mouse class I antigens utilizing antisera with a monomorphic pattern of reaction and high-resolution one-dimensional isoelectric focusing (1D-IEF). The most commonly occurring and studied H-2K and D alleles were identified in a comparison of over 40 mouse strains. By comparing H-2 mutant mouse strains, cell lines transfected with defined class I genes, or mice transgenic for a mouse class I gene and H-2 recombinant mouse strains, unambiguous identification of class I alleles was possible. The complex pattern presented by H-2-heterozygous mice was readily resolved into the contribution by the individual parental alleles. The H-2b bm series of mutants was analyzed, and for those mutants where a charge difference was predicted based on their known sequence, a change in isoelectric point (IEP) was indeed observed. Based on analysis by IEF, the bm8 mutant may contain (an) amino acid substitution(s) in addition to those reported. The present method further appears useful in elucidating defects in class I antigen synthesis and post-translational modifications, as these cannot be easily characterized when using surface staining with monoclonal antibodies (mAbs).

Alleles↗
PubMed1990

Up regulation of the Epstein-Barr virus (EBV)-encoded membrane protein LMP in the Burkitt's lymphoma line Daudi after exposure to n-butyrate and after EBV superinfection.

The Burkitt's lymphoma line Daudi carries a nontransforming Epstein-Barr virus (EBV) strain that has a deletion in the BamHI WYH region of the genome coding for the EBV nuclear antigen 2 (EBNA-2). Daudi cells fail to express the EBV-encoded latent membrane protein (LMP) (D. Ghosh and E. Kieff, J. Virol. 64:1855-1858, 1990). We show that LMP expression can be up regulated by exposure to n-butyrate and by superinfection with the B95-8 (B virus)- and P3HR1 (P virus)-derived EBV strains. Two LMP polypeptides of 60 and 48 kilodaltons (kDa) were detected in immunoblots of Daudi cells that had been exposed to 3 mM n-butyrate for 24 h. The intensity of the 48-kDa LMP increased during 72 h, in parallel with the appearance of early antigen-positive cells. The 60-kDa LMP was expressed at a low level and remained constant. Superinfection of Daudi cells with B and P virus induced the 60-kDa LMP within 3 h. In addition, P virus induced the 48-kDa LMP at a low level. The B virus-encoded EBNA-2 and EBNA-5 were detected 12 h after superinfection. The B virus-encoded 63-kDa LMP was coexpressed with the endogenous LMP after 48 h. Inactivation of the virus by UV illumination abolished the expression of the B virus-encoded antigens but did not affect the induction of the endogenous LMP. The B-cell activation marker CD23 was up regulated by B virus superinfection but not by n-butyrate exposure. CD23 was also expressed at a higher level in a stable B virus-converted subline, E95A-Daudi, that was EBNA-2 positive and coexpressed the Daudi virus- and B virus-encoded LMP. The results suggest that LMP expression is regulated by the interaction of cellular and viral factors. Binding of the virus to its membrane receptor might be involved in the triggering of cellular control mechanisms. Viral gene products are not directly involved in this function but may contribute to create a permissive cellular environment for LMP expression.

Antigens, CD↗
PubMed1990

Epstein-Barr virus (EBV) antigens processed and presented by B cells, B blasts, and macrophages trigger T-cell-mediated inhibition of EBV-induced B-cell transformation.

The ability of B cells, B blasts, and macrophages to present Epstein-Barr virion antigens to autologous T cells and trigger their capacity to inhibit Epstein-Barr virus-induced B-cell transformation was tested. Macrophages were as efficient as B cells and B blasts in presenting the virus to T lymphocytes. This function required antigen processing, because it was inhibited by chloroquine treatment and by fixation of the antigen-presenting cells immediately after viral exposure but not 18 h later. T cells exposed to the purified Epstein-Barr virus envelope antigen gp350 coupled to immunostimulating complexes also showed inhibitory function. These results suggest that recognition of processed virion antigens elicits the generation of T-cell-mediated inhibition of Epstein-Barr virus-induced B-cell transformation.

Antigens, Viral↗
PubMed1989

Relationship between clinical stage, histopathology and antibody titers against the second Epstein-Barr virus nuclear antigen (EBNA-2) in non-Hodgkin's lymphoma patients.

Non-Hodgkin lymphoma (NHL) patients with centroblastic (Cb) or centroblastic-centrocytic (Cb/Cc)-diffuse lymphomas, immunocytoma (IC) and chronic lymphocytic leukemia (CLL) in clinical stages III-IV and with active disease (highly malignant group) were compared to NHL patients with CLL, IC, and centrocytic (Cc) or centroblastic-centrocytic (Cb-Cc)-diffuse/follicular lymphomas, in clinical stages I-II and with inactive disease (low malignant group) based on the presence of antibodies to Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) and 2 (EBNA-2). In the highly malignant group, anti-EBNA-1 geometric mean titers (GMT) were 13.2 (range less than 2-80) and anti-EBNA-2 60.6 (range: 20-320). The ratio between the logarithms of anti-EBNA-1 and anti-EBNA-2 antibody titers was less than 1.0 (mean: 0.32) in all the patients examined. In 6 out of 8 patients of the low malignant group, anti-EBNA-1 titers were higher (mean: 30.1; range 10-160) than anti-EBNA-2 titers (mean: 4.3; range less than 2-80) and the EBNA 1/2 ratio was greater than 1.0. In healthy EBV-seropositive individuals, anti-EBNA-1 GMT were 49 (range: 10-320) and only 5 out of 17 individuals had detectable anti-EBNA-2 titers (GMT: 3; range less than 5-20). The EBNA-1/2 ratio was in all cases greater than 1. Among patients of the highly and low malignant groups, patients with follicular-cell-derived lymphomas had elevated antibody titers against the restricted component of early antigens (EA-R), whereas all patients with IC and 2 out of 4 CLL patients had elevated antibody titers against the diffuse component of early antigens (EA-D). The results indicate that the ratio between anti-EBNA-1 and anti-EBNA-2 antibody titers may be of diagnostic importance in patients with immunodeficiencies.

Antibodies, Viral↗
PubMed1989

Reversion of tumorigenicity and decreased agarose clonability after EBV conversion of an IgH/myc translocation-carrying BL line.

The Epstein-Barr virus (EBV)-negative Burkitt lymphoma (BL) line BL-41, and 5 independently established EBV-converted sublines, derived by infection with a transforming (B95-8) or a nontransforming (P3HR1) strain of EBV, were compared for clonability in semi-solid agarose and for tumorigenicity in immuno-suppressed mice. One P3HR1 viral convertant and 3 out of 4 B95-8 virus-converted sublines had a high (greater than 40%) agarose clonability, like the BL 41 parent, and were slightly more tumorigenic than BL-41. In contrast, the fourth B95-8 converted subline, BL-41/95, was virtually non-tumorigenic and its agarose clonability was much lower (3-23%). It showed a more drastic shift towards an LCL-like phenotype than the other convertants as reflected by high HLA class-I and EBV-encoded latent membrane protein (LMP) expression. BL 41/95 still contains the 8;14 IgH/myc translocation, carried by the parental line, and maintains the same relatively high steady-state level of c-myc mRNA and protein as the highly tumorigenic convertants. We conclude that the tumorigenicity of BL41/95 has been suppressed by a gene that acts at a level beyond the expression of the activated oncogene, in the same way as the revertants isolated from ras and SV-40-transformed cultures (Klein, 1987b; Bassin and Noda, 1987).

Animals↗
PubMed1989

Allele-specific down-regulation of MHC class I antigens in Burkitt lymphoma lines.

We have reported that Burkitt lymphomas (BL) that arise in HLA-A11 positive individuals are resistant to lysis by HLA-A11-specific and HLA-A11-restricted CTLs(10,11). Here we show that this phenomenon can be explained by a selective loss of the HLA-A11 polypeptide. The HLA-A11 negative phenotype is due to a regulatory phenomenon, rather than a structural defect, as proven by the ability to rescue expression of HLA-A11 in in vitro Epstein-Barr virus (EBV)-converted sublines of EBV negative BLs.

Alleles↗
PubMed1989

The role of methylation in the phenotype-dependent modulation of Epstein-Barr nuclear antigen 2 and latent membrane protein genes in cells latently infected with Epstein-Barr virus.

Seven virus-encoded proteins are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid (LCL) cell lines: the EBV nuclear antigens EBNA 1 to 6 and the latent membrane protein (LMP). In nasopharyngeal carcinoma (NPC), only EBNA 1 is regularly expressed; LMP is detected in about 50% of the tumours. In Burkitt's lymphoma (BL) tumours, only EBNA 1 is expressed. Also, in BL-derived cell lines that maintain the phenotypic markers characteristic of the in vivo tumour (group I), only EBNA 1 is expressed. EBV was rescued by induction or cocultivation from one BL cell line with a restricted group I pattern, and from one NPC tumour, into normal B cells. In the resulting LCLs EBNA 1 to 6 and LMP were expressed. We assessed the level of methylation in the genes encoding ENBNA 2 and LMP by restriction fragment analysis using the methylation-sensitive enzymes SmaI and HpaII. These genes were extensively methylated in the group I BL line Rael and the nude mouse-passaged C15 NPC tumour, but were demethylated in the derived LCLs. In the LMP-expressing NPC C15 tumour the 5' flanking region of the gene was hypomethylated, whereas the coding exons were methylated [corrected]. The EBNA 1 coding exon was methylated in the Rael line and in NPC, in spite of expression. In contrast, CpG pairs in oriP were originally hypomethylated and remained so after their transfer to LCLs. The cell phenotype-dependent pattern of EBV gene methylation correlated with the phenotype-dependent pattern of EBNA and LMP expression. The specific patterns of methylation localized to controlling regions (oriP and 5' flanking sequences) also suggest a specific role for methylation in the regulation of EBNA and LMP expression.

Antigens, Viral↗