Epstein-Barr Virus Sequences Research Articles

Frequent c-myc Oncogene Activation and Infrequent Presence of Epstein-Barr Virus Genome in AIDS-Associated Lymphoma

Abstract Sixteen cases of histologic intermediate-grade and high-grade AIDS- associated non-Hodgkin's lymphoma (NHL) were studied for the presence and patterns of c-myc gene and bcl-2 locus rearrangements. The presence of Epstein-Barr virus (EBV) sequences and proteins and HTLV-I sequences were also investigated. c-myc gene rearrangements analogous to those observed in sporadic Burkitt lymphomas were detected in 12 of 16 cases. Six of 16 cases had detectable EBV sequences and proteins. None of the cases displayed bcl-2 rearrangements or contained HTLV-I sequences. These data suggest a frequent role for c-myc activation in the pathogenesis of AIDS-associated NHL, independent of histologic type. Conversely, EBV does not appear to be directly involved in lymphomagenesis in the majority of AIDS-associated NHLs.

Human neonatal lymphocytes immortalized after microinjection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV) is a highly efficient acute transforming agent in human cells, provided that the intact virus is used. To investigate the ability of viral DNA alone to transform cells, we introduced the EBV genome into human lymphocytes. After microinjection of EBV DNA into neonatal B lymphocytes, we established a cell line that in early passages contained multiple viral fragments. This cell line retained sequences from the short, unique (Us) region of the EBV genome and sequences from EcoRI-E. The viral sequences were not expressed; however, the cells expressed a 2.3-kilobase polyadenylated message homologous to the c-fgr oncogene, a cellular locus believed to be activated by EBV infection [M. S. C. Cheah, T. J. Ley, S. R. Tronick, and K. C. Robbins, Nature (London) 319:238-240.]. The cell line was monoclonal with rearrangement at the immunoglobulin locus and had a reciprocal translocation t(1;7)(p34;q34) and a deletion of sequences within the locus for the beta chain of the T-cell receptor. The close proximity of the translocation to the chromosomal loci for c-fgr on chromosome 1 and the T-cell receptor beta chain on chromosome 7 suggests that structural alteration of these genes was critical to this transformation event.

Localization polymorphism of EBV DNA genomes in the chromosomes of Burkitt lymphoma cell lines

The localization of Epstein-Barr virus (EBV) genomes in nuclei of the human lymphoblastoid cell lines Raji, Jijoye, P3HR-1, Daudi and Ramos was investigated by in situ hybridization with biotinylated EBV DNA probes. We found that all sites of hybridization were associated with the chromosomes. Only some of these sites were present on both chromatids and these had a non-random distribution; these sites could represent EBV sequences integrated at specific points on the chromosomes. The total mean site number corresponded with the number of viral DNA copies estimated in the different cell lines by other techniques, but the copy number was highly variable from cell to cell in a given line.

The properties and sequence of glycoprotein H of herpes simplex virus type 1

The map position of the coding sequence of glycoprotein H of herpes simplex virus type 1 was determined by marker transfer studies in which DNA fragments cloned from a virus resistant to neutralisation by an anti-gH monoclonal antibody were used to transfer antibody resistance to wild type virus DNA following cotransfection. The gH coding sequence was mapped to the BglII “m” fragment of HSV-1 DNA (map coordinates 0.27–0.312), confirming the map position previously determined by intertypic recombinant analysis ( Buckmaster et al., 1984). The complete nucleotide sequence of the BglII “m” fragment revealed two large open reading frames in addition to the thymidine kinase gene. The open reading frame lying immediately 3′ of the thymidine kinase gene has a predicted translation product with the features of a large glycoprotein. This open reading frame translates to an amino acid sequence of 90,323 mol wt with a signal peptide, a membrane anchor sequence, a large external domain containing potential N-glycosylation sites, and a charged C- terminal cytoplasmic domain. We suppose that this amino acid sequence corresponds to gH of HSV-1, and A. Davison (personal communication) has noted the existence of homologous glycoproteins predicted from the nucleotide sequences of Varicella-zoster virus and Epstein-Barr virus. The properties of monoclonal antibody LP11, directed against gH show remarkable similarities to the properties for gD antibodies. LP11 efficiently neutralises virus infectivity, blocks cell fusion by syncytial virus strains, and inhibits the formation of plaques when added to cell monolayers after infection. These similarities in antibody activity imply functional relatedness between gH and gD of herpes simplex virus.

EVIDENCE THAT RESPIRATORY TRACT IS MAJOR RESERVOIR FOR EPSTEIN-BARR VIRUS

Exfoliated cells harvested from bronchial washings of 53 patients with suspected bronchogenic carcinoma were tested by means of DNA dot hybridisation using the cloned large internal repeat (IR) sequence of Epstein-Barr virus (EBV) genome as a probe. 25 of these patients gave positive results. Since the patients had diseases that were not related to the virus, this finding suggests that the lower respiratory tract is a major reservoir for EBV. Attempts at cellular localisation of the virus revealed only an occasional cell which harboured the viral genome or expressed viral capsid antigens. These cells could not account for the quantity of the viral DNA detected in bronchial washings. Moreover, patients had similar profiles of serum EBV antibodies whether they were positive or negative for EBV DNA by dot hybridisation. These findings are compatible with a state of viral latency in which cells harbour a low copy number of the viral genome. Viral expression rarely occurs in these cells, which seem to elicit a minimum host immune response. If it is assumed that each latently infected cell harbours a maximum of approximately 30 EBV genomes (which is the lower limit of detection by the

Identification of Epstein-Barr virus sequences that encode a nuclear antigen expressed in latently infected lymphocytes.

The Epstein-Barr virus (EBV) BamHI restriction endonuclease fragment K (B95-8 strain) was introduced into a polyoma virus expression vector and used to transfect murine NIH 3T3 cells. An EBV-associated nuclear antigen was detected in these cells in an indirect immunofluorescence test using anti-EBV nuclear antigen-positive human sera. These sera recognized a Mr 88,000 polypeptide in 3T3 cells transfected with the BamHI fragment K-containing polyoma virus plasmid by radioimmunoelectrophoresis. A Mr 88,000 polypeptide also was detected in a B-cell line latently infected with the B95-8 strain of EBV. Plasmids containing insertion and deletion mutations in BamHI fragment K directed the synthesis of truncated forms of the Mr 88,000 polypeptide in 3T3 cells. These data directly demonstrate that the polypeptide identified in EBV-infected lymphocyte lines by anti-EBV nuclear antigen-positive human sera is encoded by the viral genome.

Epstein-Barr virus with heterogeneous DNA disrupts latency.

By cloning the HR-1 Burkitt lymphoma line, we previously uncovered two distinct biological variants of nontransforming Epstein-Barr virus (EBV). The most commonly cloned variant has a low rate of spontaneous viral synthesis and is unable to induce early antigen in Raji cells (EAI-). A rare variant spontaneously releases virus which is capable of inducing early antigen in Raji cells (EAI+). Since EAI- virus lacks heterogeneous DNA (het-) and EAI+ virus contains heterogeneous DNA (het+), we suggested that spontaneous viral synthesis and induction of early antigen are biological properties which correlate with the presence of het sequences. The present experiments provide three new lines of experimental evidence in favor of this hypothesis. (i) Revertant subclones of the EAI+ het+ variant which have lost the het DNA concomitantly lost EAI ability. Thus, het DNA is not stably associated with the cells as are the episomes. (ii) het DNA was acquired by two het- subclones of the HR-1 line after superinfection with EAI+ virus. After superinfection, these clones synthesized EAI+ het+ virus. Thus, het DNA may be maintained in the HR-1 line by cell-to-cell spread. (iii) Virus with het DNA activated full expression of endogenous latent EBV of the transforming phenotype in a line of immortalized neonatal lymphocytes designated X50-7. By use of restriction endonuclease polymorphisms unique to both the superinfecting and endogenous genomes, we show that the genome of the activated virus resembles that of the virus which was endogenous to X50-7 cells. This result suggests that het sequences result in transactivation of the latent EBV. het DNA had homology with EBV sequences which are not normally contiguous on the physical map of the genome. het DNA was always accompanied by the presence of DNA of nonheterogenous HR-1. Thus, het DNA is a form of "defective" EBV DNA. However, the biological effect of this defective DNA is to enhance rather than to interfere with EBV replication. This is a novel property of defective virus.

The EB virus genome in Daudi Burkitt's lymphoma cells has a deletion similar to that observed in a non-transforming strain (P3HR-1) of the virus.

Epstein-Barr virus (EBV) DNA isolated from the frequently studied and unusual Burkitt's lymphoma cell line, Daudi, contains a 7.4-kb deletion, similar to (but larger than) that found in a non-transforming isolate of the virus, P3HR-1. A comparison of EBV sequence in Daudi cells with that from a comparable region in a wild-type, transforming strain of the virus (B95-8) indicates that at least two of the previously identified RNAs, a highly repetitive sequence, and other interesting coding or structural features should be absent in Daudi EBV DNA as a consequence of the deletion. The information removed by the deletion, as well as that which might be generated by juxtaposition of two regions of the genome that are not adjacent in most strains of the virus are discussed.

One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain.

A gene fusion between an Epstein-Barr virus (EBV) triplet nucleotide repeat array (IR3), which has homology to host DNA, and lacZ was used to demonstrate that this EBV sequence encodes part of the Epstein-Barr nuclear antigen (EBNA). The IR3 sequence is translated into a glycine-alanine copolymer that reacts with anti-EBNA human sera. Some EBV-immune human antisera recognize a second intranuclear protein that is also specific for latently infected cells and is designated EBNA2. EBNA2 is not related to EBNA1 because the molecular mass of EBNA2 is 82 kilodaltons, whereas that of EBNA1 varies from 68 to 85 kilodaltons among cells transformed by different EBV isolates; also EBNA2 does not contain the copolymer domain of EBNA1.

Comparative evaluation of various techniques to detect EBV DNA in exfoliated nasopharyngeal cells

Summary Exfoliated epithelial cells from nasopharynx of 27 individuals were collected with a low-negative-pressure suction apparatus. These cells were examined cytologically and stained for Epstein-Barr virus (EBV) nuclear antigen on touch smears by immunoperoxidase. DNA was extracted from the remaining cells and studied for the presence of EBV sequences by blot and spot hybridization. In the 22 positive nasopharyngeal carcinoma cases (confirmed by clinical, histological and cytological examination) EBV DNA was found with both techniques in 21 cases, while in the 5 cases of chronic inflammation without tumorous cells, no EBV DNA was detected.

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

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