Epstein Barr virus (EBV)-carrying cells of a chronic lymphocytic leukemia (CLL) subpopulation express EBNA1 and LMPS but not EBNA2 in vivo.
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Antigens, Viral↗
Biomedical subjects
I Ernberg
Publications and source records attributed to I Ernberg.
Loss of heterozygosity on chromosome arm 3p in nasopharyngeal carcinoma.
We have examined 17 primary undifferentiated nasopharyngeal carcinoma biopsies for allelic loss on 3p, comparing the findings in tumors with those in normal lymphocyte DNA from the same patients. Ten polymorphic microsatellite markers were used between 3p13 and 3p26. Allelic loss was observed in 12 samples (70%). Two loci were most frequently affected: D3S1067 (3p21.1-14.3) in 60% and D3S1217 (3p14.2-14.1) in 58%. One tumor seemed to have a homozygous deletion at 3p26, detected by the D3S1297 marker. Analysis of the clinical data showed that an increased number of aberrations in 3p was correlated with more advanced tumor stages.
Adult↗
Epstein Barr virus (EBV)-carrying cells of a chronic lymphocytic leukemia (CLL) subpopulation express EBNA1 and LMPs but not EBNA2 in vivo.
We have previously described an exceptional CLL patient, P.G., whose leukemic cell population contained a small fraction of Epstein-Barr virus (EBV)-carrying cells. These cells grow directly into permanent cell lines in vitro. Using RT-PCR analysis, we now show that the in vivo EBV-carrying CLL cells expressed EBNAI, LMPI, LMP2a and 2b, but not EBNA2, in 4 of 4 blood samples obtained during the last 3 years of the patient's life. Our data also show that the CLL cells used a promoter in the F/Q, but not the W or C, region. This is consistent with the fact that CLL cells resemble resting lymphocytes rather than immunoblasts. Expression of LMP1 and LMP2b differs from the exclusive EBNAI and LMP2a expression of normal resting B cells, however, and corresponds to the state defined as latency II. This form of latency was until now detected only in EBV-carrying non-B cells in vivo. Our data show that a B-cell subtype can also show this expression pattern in vivo.
Antigens, Viral↗
Synthetic peptides deduced from the amino acid sequence of Epstein-Barr virus nuclear antigen 6 (EBNA 6): antigenic properties, production of monoreactive reagents, and analysis of antibody responses in man.
Studies on the antibody responses to various Epstein-Barr virus (EBV) antigens have been instrumental in the understanding of the seroepidemiology and diagnosis of this viral infection and the subsequent carrier state. While antibodies to the viral capsid antigen (VCA), early antigen (EA), and nuclear antigens 1 and 2 (EBNA 1 and 2) have been well characterized, the antibody response to the other nuclear antigens is not well understood. EBNA 6 is expressed by lymphoblasts during acute EBV infection and may be an important antigen for diagnosis and evaluation of the immune response. In order to analyze the antibody response to EBNA 6, ten peptides (20-21 amino acids), deduced from the EBNA 6 coding region, were synthesized and evaluated for antigenicity by ELISA. One peptide (p-63; PAPQAPYQGYQEPPAPQAPY) derived from the amino acid repeats showed the highest specific reactivity with human sera. This peptide was evaluated further for detection of human EBNA 6-reactive antibodies. Forty-two of forty-nine (86%) EBV-seropositive healthy donors had p-63-specific IgG reactivity, while none of 50 EBV-seronegative patients reacted with the p-63 peptide. Twenty-two of fifty-one (43%) patients with ongoing primary EBV infection had detectable p-63-specific IgG. Serum samples drawn sequentially from patients during and after primary EBV infection revealed an increase in p-63-reactive IgG over time.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Differences in the growth pattern and clinical course of EBV-LMP1 expressing and non-expressing nasopharyngeal carcinomas.
All low differentiated or anaplastic forms of nasopharyngeal carcinoma (NPC) carry multiple copies of EBV-DNA and express EBNA1. The major membrane protein, LMP1, is only expressed in 65% of the tumours. The physiological function of LMP1 in the viral life cycle is unknown, but it has been shown to transform established rodent fibroblasts and immortalised human keratinocytes in vitro, and to increase the likelihood of a malignant transformation. We studied 74 cases collected from the Shanghai and Guanzhou areas in China. LMP1 expression was assessed in tumour biopsies by immunoblotting. Clinical and follow-up data were evaluated according to the classification of WHO. The laboratory and the clinical data were assembled in a mutually independent double blind fashion. Our findings indicate that the LMP1-positive tumours grew faster and more expansively than LMP1-negative tumours, but nevertheless had a better prognosis. LMP1-negative tumours recurred at a higher frequency, and showed an increased tendency to metastasise.
Carcinoma↗
Coupled transcription of Epstein-Barr virus latent membrane protein (LMP)-1 and LMP-2B genes in nasopharyngeal carcinomas.
The presence of transcripts of the Epstein-Barr virus genes for Epstein-Barr nuclear antigen (EBNA)-1 and EBNA-2 and for latent membrane protein (LMP)-1, LMP-2A and LMP-2B was investigated in 24 nasopharyngeal carcinoma (NPC) biopsies of Chinese origin and two NPC-derived solid tumour lines, CAO and C15, of Chinese and north African origin respectively, propagated by serial transplantation in nude mice. Transcripts were detected by PCR amplification of cDNA. EBNA-1 transcripts were present in all biopsies tested and originated exclusively from the FQ promoter while the C and W promoters were inactive. Using nested primers, LMP-1 and LMP-2B RNAs were found to be co-ordinately expressed in 22 of the 24 biopsies, while the two remaining tumours were negative for both. LMP-2A transcription was detected in 17 of the 22 LMP-1-positive biopsies. In summary, the following patterns of viral gene expression were observed in the tumour biopsies: (i) LMP-1-LMP-2B and LMP-2A positive (17 biopsies); (ii) LMP-1-LMP-2B positive but LMP-2A negative (five biopsies); (iii) no viral gene other than EBNA-1 expressed (two biopsies).
Animals↗
The role of repetitive DNA sequences in the size variation of Epstein-Barr virus (EBV) nuclear antigens, and the identification of different EBV isolates using RFLP and PCR analysis.
The six Epstein-Barr virus (EBV) nuclear antigen proteins (EBNA-1-6) show characteristic size variations between different virus isolates; this is a feature that has been used to identify the source of virus isolates in epidemiological studies (Ebnotyping). We have now studied the correlation between restriction fragment length polymorphisms (RFLPs) within exons coding for the EBNAs and the molecular masses of the respective proteins. The B95-8 EBV strain was used as the prototype virus. The variation in apparent molecular mass of EBNA-1, -3 and -6 correlated positively with the size of RFLP coding for repeat sequences in these polypeptides. For EBNA-2, no correlation between apparent molecular mass and length of the repetitive sequences was found. The EBNA-4 protein showed virtually no variation in apparent molecular mass and RFLP size across the repeat sequence. Based on the strong correlation between apparent molecular mass and RFLP size for EBNA-6, we developed an EBNA-6 PCR assay that discriminated between different isolates of EBV. This assay offers the advantage of EBV characterization using uncultured material (e.g. throat washings, blood or biopsies), thus avoiding the selection against poorly transforming strains that occurs during establishment of lymphoblastoid cell lines required for Ebnotyping at the protein level.
Animals↗
A subpopulation of normal B cells latently infected with Epstein-Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1.
Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.
Animals↗
Upregulated local cytokine production in recurrent tonsillitis compared with tonsillar hypertrophy.
In children with recurrent tonsillitis there may be persistent antigen deposition in tonsil tissue. even between exacerbations. If so, upregulation of immunocompetent cells should occur continuously, in contrast to tonsil tissue from children with tonsillar hypertrophy. The cytokine pattern was studied in cell suspensions prepared from tonsils obtained from 12 children undergoing tonsillectomy. The study group comprised 6 children with recurrent tonsillitis and 6 who had a history of tonsillar hypertrophy causing sleep apnea. Cytokine-producing cells (IL-1alpha, IL-1beta, TNFalpha, IL-6, IL-8, IL-2, IFNgamma, TNFbeta, IL-10 and IL-4) were characterized at the single-cell level by use of cytokine-specific monoclonal antibodies and indirect immunofluorescence technique. A constitutive production of IL-1alpha, IL-1beta, TNFalpha, and IL-8 was found in both groups (10-300/10(5) cells). However, the frequency of spontaneous IL-2, IFNgamma, TNFalpha, IL-6 and IL-10 was consistently low (10 +/- 10 cells) in both groups. Following restimulation by T-cell receptor ligation, using immobilized anti-CD3 mAb, with concentrations chosen so that it did not activate resting cells, increased frequencies of TNFalpha, IL-6, IL-8, IL-2, IFNgamma, IL-4 and 1L-10 synthesizing cells were induced in the recurrent tonsillitis group. Significantly higher incidences of IL-1beta, IL-6 and IL-2 producing cells were found in the recurrent tonsillitis group (60-200/10(5) cells, p <0.05). Microbiological evaluation in the tonsil tissue could not reveal tiny differences between the studied groups regarding bacterial or viral pathogens. However, this does not exclude persistent increased intracellular deposition of microbial antigens as a possible explanation for the elevated incidence of IL-1beta, TNF-alpha, IL-6, IL-8, IL-2, IFNgamma, IL-10 and IL-4 expressing cells noticed in patients with recurrent tonsillitis.
Cells, Cultured↗
Molecular epidemiology of Epstein-Barr virus infection.
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Biological Evolution↗
DNA of lymphoma-associated herpesvirus (HVMF1) in SIV-infected monkeys (Macaca fascicularis) shows homologies to EBNA-1, -2 and -5 genes.
We have characterized a new Epstein-Barr-virus(EBV)-like herpesvirus associated with lymphomas of SIV-infected cynomolgus (Macaca fascicularis) monkeys and propose that this virus is designated herpesvirus macaca fascicularis I (HVMFI). Genomic regions in HVMF1 of potential significance for tumor pathogenesis were analyzed by Southern blotting, PCR and sequencing, and compared with human EBV DNA. Virus from 7 SIV-associated lymphomas and one lymphoma-derived cell line were shown to share homology with the EBNA1- and EBNA2-coding regions of EBV, while some homology to EBV-LMP1 was detectable only at low-stringency hybridization. Homologous regions to the long internal repeat (IR1; BamHI W), the EBER1 and 2 and the latent origin of DNA replication (oriP) could also be demonstrated in HVMF1. These coding regions, except IR1, showed restriction-enzyme maps different from those of EBV. Sequencing of the EBNA5 homologous region of HVMF1 DNA, corresponding to exons W1 and W2, showed 65% homology to the EBV exons W1 and W2, and 80% to the whole region including the intron. Since EBNA5 has been reported to bind tumor-suppressor proteins p53 and Rb in vitro, the HVMF1 homology could be important for the lymphomagenic capacity of this monkey herpesvirus.
Amino Acid Sequence↗
Sequence-specific methylation inhibits the activity of the Epstein-Barr virus LMP 1 and BCR2 enhancer-promoter regions.
We reported earlier that variable expression of the Epstein-Barr virus (EBV) encoded membrane protein LMP 1 in nasopharyngeal carcinoma and the host-cell-phenotype-dependent activity of the BCR2 promoter (one of the possible initiator sites for transcripts of Epstein-Barr nuclear antigens) in Burkitt's lymphoma (BL) lines can be related to the methylation status of the 5'-flanking regulatory regions of the BNLF 1 and BCR2 promoter, respectively. Here we report that clones of the BL line Mutu that differ in expression of LMP 1 also show a differential methylation pattern of the LMP 1 regulatory sequences: this region is hypomethylated in an LMP 1 expressing (group III) clone but methylated in a group I clone that does not express LMP 1. We introduced in vitro methylated reporter plasmids carrying BNLF 1 and BCR2 enhancer-promoter sequences into the BL line Raji and found that overall methylation of 5'-CG-3' sequences by the Spiroplasma methylase Sssl significantly reduced their activity compared to unmethylated or mock-methylated controls. Methylation of 5-CCGG-3' sequences by Hpall methyltransferase gave similar results. On the contrary, methylation of 5'GCGC-3' sequences by Hhall methyltransferase gave similar results. On the contrary, methylation of 5'-GCGC-3' sequences by Hpal methyltransferase resulted only in a moderate reduction of BNLF 1 enhancer-promoter activity. These data support the notion that methylation at discrete sites within control regions of latent, growth-transformation associated EBV genes may contribute to silencing their expression.
Burkitt Lymphoma↗
Epstein-Barr virus: adaptation to a life within the immune system.
Epstein-Barr virus has developed multiple strategies to ensure its long-term persistence in the infected B cells of immunocompetent hosts. These include the establishment of cell-phenotype-specific programs of viral gene expression and the transduction of cellular genes that modulate immune responses. Cytotoxic T cells may specifically influence the evolution of this genetically stable virus.
Adaptation, Physiological↗
Detection of multiple 'Ebnotypes' in individual Epstein-Barr virus carriers following lymphocyte transformation by virus derived from peripheral blood and oropharynx.
Transformation of a B lymphocyte into a lymphoblastoid cell line (LCL) by Epstein-Barr virus (EBV) results in the expression of EBV nuclear antigens (EBNAs) of which the size spectrum ('Ebnotype') is characteristic for the transforming virion. Ebnotyping has been used as an epidemiological tool for studies of EBV infection. We compared the occurrence of a single and of multiple Ebnotypes, as defined by EBNAs 1, 2 and 6, in healthy and diseased EBV carriers. Cases from which two or more LCLs could be established from peripheral blood or oropharyngeal cultures were considered informative. The frequency of multiple Ebnotypes was relatively low in healthy individuals and in patients with infectious mononucleosis or with haematological diseases who were awaiting a bone marrow transplant [blood, 11 of 74 patients (15%); oropharynx, 12 of 49 patients (24%)], whereas it was relatively high in recipients of bone marrow or cardiac allografts and one patient with AIDS [blood, 12 of 34 patients (35%); oropharynx, 11 of 16 patients (69%)]. Three patterns of the simultaneous presence of multiple Ebnotypes were distinguished. The first, most frequent, pattern observed predominantly in oropharyngeal cultures of all groups consisted of minority Ebnotypes differing from the majority type by only a single EBNA protein (usually EBNA 1). The second, less frequent, pattern observed in the healthy carriers and the (candidate) transplant recipients consisted of minority Ebnotypes differing from the majority type by two EBNA proteins (mostly EBNAs 1 and 6). The third pattern, characterized by the simultaneous presence of totally different Ebnotypes, was restricted to the (candidate) transplant recipients and the AIDS patient and was more frequently observed in the blood than in the oropharynx. We suggest that the first two patterns result from heterologous recombinations occurring during viral replication at repeat sequences within the EBNA coding regions, whereas the third pattern reflects multiple infections with exogenous viruses.
Acquired Immunodeficiency Syndrome↗
Expression of Epstein-Barr-virus-related nuclear antigens and B-cell markers in lymphomas of SIV-immunosuppressed monkeys.
Simian-immunodeficiency-virus(SIV)-infected cynomolgus monkeys develop B-cell lymphomas in approximately one third of the cases. We have now studied the expression of cynomolgus-Epstein-Barr-virus(cyno-EBV) nuclear antigens in 13 cyno-EBV-carrying SIVsm-associated monkey lymphomas and established cell lines from 3 of these tumors. Immunoblots of cell lysates were probed with polyspecific and monospecific reagents directed against human EB-virus EBNAI-6, and against the membrane protein LMPI. An EBNA2-cross-reacting protein was demonstrated in 8 lymphoma tissues (8/13) and in the 3 cell lines derived from the tumors. All tumors expressed a polypeptide with 50 to 55 kDa molecular weight, which cross-reacted with some antibodies to EBNAI. Absorption experiments with normal monkey tissue showed that this polypeptide was specific for the cyno-EBV-carrying lymphoma cells. Equivalents of EBNA3-6 and LMPI could not be detected. Immunophenotypical characterization showed that the monkey lymphomas were similar to human HIV-associated B-cell lymphomas. Malignant B-cell lymphomas in experimentally SIVsm-infected cynomolgus monkeys can be a model for EBV-associated lymphomagenesis in immunodeficiency states.
Animals↗
An origin of DNA replication (oriP) in highly methylated episomal Epstein-Barr virus DNA localizes to a 4.5-kb unmethylated region.
Epstein-Barr virus (EBV) reveals two types of interactions with human B lymphocytes. In immortalized lymphoblastoid cell lines (LCL), nine virus-coded proteins are expressed: six nuclear (EBNA1-6) and three membrane proteins (LMP1, TP 1,2). In Burkitt's lymphoma (BL) tumors and representative cell lines (type I) only EBNA-1 is expressed. The viral genomes are carried by the cells in an extrachromosal, episomal form. We have earlier described an overall difference in EBV DNA methylation between LCLs of normal B-cell origin and type I BL lines. EBV genomes are hypomethylated in the former, methylated in the latter. The origin of replication (oriP) for the episomal DNA is exempted from this overall methylation. This unmethylated region was analyzed in the BL derived type I cell line Rael. It starts just upstream of the coding exons for the small untranslated RNAs EBER I/II. It extends throughout oriP and includes several control elements between oriP and the first promoter for EBNA transcription (BCR2). The viral DNA was methylated around and downstream of that promoter, which is not used for EBNA-1 expression in Rael.
Animals↗
Clonability and tumorigenicity of human epithelial cells expressing the EBV encoded membrane protein LMP1.
Two isolates of the EBV-LMP1 gene were compared for their ability to induce phenotypic changes in a non-tumorigenic human keratinocyte line, Rhek-1, immortalized with an adenovirus 12-SV40 hybrid virus. One isolate, designated B-LMP1, was derived from B95-8, a B-cell line of marmoset origin, that carries a viral strain from a mononucleosis patient. The other, designated C-LMP1, originated from a nude mouse passaged Chinese NPC tumor, CAO. Both types of transfectants were less serum dependent than the non-transfected and the vector-transfected controls. The ability to grow on low serum increased with increasing LMP1 expression. All transfectants were more highly clonable than the non-transfected or vector-transfected controls. Clonability in soft agarose increased with increasing LMP1 expression. Nine of 24 C-LMP1 transfectants produced tumors in SCID mice. Seven of them grew invasively into the surrounding tissue. Only one of 12 B-LMP1 transfected Rhek-1 clones was tumorigenic. It did not grow invasively. All tumorigenic transfectants expressed LMP1 at high or moderate levels. All tumors were found to express LMP1. Transfectants with low LMP1 expression did not produce tumors. The untransfected Rhek-1 cells and six vector control clones failed to produce tumors.
Adenoviridae↗
A monkey model for Epstein Barr virus-associated lymphomagenesis in human acquired immunodeficiency syndrome.
High-grade malignant nonHodgkin's lymphomas--five lymphoblastic, three pleomorphic, and two immunoblastic--developed in 10/25 cynomolgus monkeys (Macaca fascicularis) followed for up to 746 d after infection with simian immunodeficiency virus, strain SIVsm. These lymphomas were shown to be associated with an Epstein-Barr (EB)-like cynomolgus B-lymphotropic herpesvirus (CBLV) by electron microscopy, by Southern blot hybridization with probes against human EBV, and by the expression of antigens corresponding to EBV-associated nuclear antigens (EBNAs) involved in human B cells transformation. Southern blot demonstration of immunoglobulin gene rearrangements and homogeneous EBV episomes indicated that all the lymphomas were CBLV-associated monoclonal B cell proliferations. Our findings suggest that these tumors correspond to the EBV-associated malignant lymphomas in acquired immunodeficiency syndrome with respect to clinical, morphological, phenotypic, and genotypic characteristics. The particular susceptibility of SIVsm immunodeficient cynomolgus monkeys for CBLV-associated lymphomagenesis appears therefore a useful model for EBV-associated lymphomas in humans.
Animals↗