We have described 5 major subtypes of Epstein-Barr virus (EBV) based on variations in EBNA-1 sequences. These include P-ala (identical to the prototype B95.8 virus), P-thr, V-pro, V-leu, and V-val. Normal individuals often carry multiple EBV subtypes, the most common being P-ala, whereas EBV-associated tumors examined to date always contain a single subtype, which only on rare occasion is P-ala. The primary hypotheses that these observations generate are as follows: (1) Each of these EBV subtypes are naturally occurring, and in normal individuals the multiplicity of subtypes results from multiple infections. (2) EBV subtypes in normal individuals are generated in vivo from a single infecting virus subtype by mutations in EBNA-1. The second hypothesis essentially excludes the possibilities that the nonrandom association of certain subtypes with lymphomas is secondary to the geographic distribution of EBV subtypes and, if proven correct, could provide strong support for a direct role of EBV in tumorigenesis. In this report, we provide evidence for the latter hypothesis. We show that the P-ala EBV subtype present in most nasal lymphomas undergoes and accumulates multiple mutations consistent with the generation of variant species of EBNA-1 in vivo. This phenomenon is similar to the generation of quasispecies in RNA viruses and is the first description of in vivo generation of subtypes in DNA viruses. In RNA-based viruses, including human immunodeficiency virus and hepatitis C virus, the emergence of quasispecies is linked to replication infidelity and significantly influences disease processes through its effect on viral tropism, the emergence of viruses resistant to the host defenses or to therapy, and pathogenicity. The present data thus raise important questions relating to the mechanisms whereby these mutations are generated in EBV and their relevance to the pathogenicity of EBV-associated lymphomas.

In seropositive individuals Epstein-Barr virus (EBV) establishes a virus reservoir in peripheral blood lymphocytes (PBLs). Transmission from one individual to another occurs via saliva due to a lytic (virion productive) phase of infection in the oropharynx. EBNA-1 is responsible for maintaining viral episomes in the host cell and could, therefore, also affect the persistence of the virus in different cell lineages. Based on sequence analysis of EBNA-1 we now demonstrate that (i) in addition to the prototype EBNA-1 (identical to the B95.8 virus EBNA-1), EBV in normal individuals encompasses multiple EBNA-1 subtypes, both in PBLs and in oral secretions; (ii) although EBV with prototype EBNA-1 is the predominant virus in normal individuals, it is very rarely associated with either nasopharyngeal carcinoma (NPC) or Burkitt's lymphoma (BL); (iii) EBV with an EBNA-1 subtype (V-val) frequently associated with NPC is also selectively detected in oral secretions and not in PBLs; (iv) EBV with the EBNA-1 subtype V-pro is restricted to PBLs, while a mutated version of this subtype is present in BL, but not in NPC. These findings suggest that the variations in EBNA-1 may be relevant to the ability of EBV to persist in different cell types, and hence relevant to its oncogenic potential.

To target expression of toxic genes to Epstein-Barr virus (EBV)-associated tumor cells, we have developed an EBV-driven enzyme prodrug system (EDEPS) that takes advantage of the trans-activating properties of EBNA1, a latent protein expressed in all EBV-containing cells, to direct expression of cytosine deaminase (CD) at high levels in those cells only, Plasmids were constructed in which the CD gene or a luciferase reporter gene were cloned downstream of the herpes simplex virus thymidine kinase (tk) promoter and the family of repeats (FR) sequence from the oriP region of EBV, Analysis of luciferase activity after transient transfection into a panel of EBV-negative or -positive human cell lines showed that the presence of the FR element enhanced transcription from the tk promoter in all EBV-positive cell lines, whereas transcription from tk was repressed in all EBV-negative cell lines, including B, T, and fibroblast cell lines, In clonogenicity assays following transfection with the CD vector, the presence of 5-fluorocytosine (5-FC) in the culture medium completely abolished cell growth in EBV-positive cell lines, but did not affect the growth of EBV-negative cell lines, This vector system should have wide applicability in that it allows targeted expression of any gene of interest to tumors that carry EBV, irrespective of the role EBV plays in their pathogenesis.

We have examined sequence variations in the EBNA-1 protein of EBV in normal peripheral blood lymphocytes (PBL) and Burkitt's lymphomas (BL), We find two EBNA-1 strains P (prototype) and V (variant) which differ by 15 amino acids, Each strain has two subtypes defined by the amino acid at position 487 (P-ala, P-thr, V-pro and V-leu), In PBLs from 32 normal individuals, up to three of these subtypes were found in each sample, but the V strain did not occur in the absence of P strain viruses, nor was the V-leu subtype ever observed in normal PBL, In BLs only a single subtype was observed in each tumor, The P-thr and V-leu subtypes were more frequently seen than the P-ala and V-pro subtypes, which occurred in only two and one of the 36 tumor samples respectively, The P-thr was the most commonly observed peripheral blood of both American and as well as in African tumors, However, in 11 of 12 American tumors, the EBNA-1 subtype was V-leu, These data indicate that some EBNA-1 subtypes are more likely to lead to oncogenesis, and one subtype, V-leu, appears only to occur in tumors.

The growth arrest mediated by p53 is caused at least in part by the p53 mediated expression of p21 (p21(waf1/Cip1)). Since only one-third of primary Burkitt's lymphomas (BL) demonstrate mutations in the p53 gene, we examined the structural integrity of the p21 coding region by single-strand conformational polymorphism and DNA sequence analysis to determine the extent to which this gene is mutated in BL. Of 34 BLs analyzed, a frequent change (38%) at codon 31 that replaced Ser with Arg was found in 13 samples, 10 of which were from Africa. This change at codon 31 is also detected in peripheral blood DNA from normal subjects and may thus represent a polymorphism. One BL cell line, DH978, carried a change at codon 63: Phe to Leu. This mutation was heterozygous, and both the wild-type and the mutated p21 mRNA were expressed in the tumor cell Line. By transfection experiments, the mutant p21 was less efficient in suppressing clonogenicity than wild-type p21. To our knowledge, this is the only mutation described in p21. The availability of this mutant p21 should further help in functional studies of p21.

Constitutive expression of c-myc resulting from a chromosomal translocation, which juxtaposes c-myc to an immunoglobulin gene, is a pivotal lesion in Burkitt's lymphomas. This deregulated expression of c-myc is associated with mutations in the regulatory regions, i.e. the first exon and the first intron of c-myc in tumors where the chromosomal breakpoint is not itself within the regulatory region. Until recently it was widely believed that the c-myc protein in these tumors is wild type. We have demonstrated that in a fraction of Burkitt's lymphomas from Africa and from the continental USA, and in mouse plasmacytomas, the c-myc gene carries mutations in the coding region. We now show that, occasionally, such mutations are also present in multiple myelomas--tumors which do not carry translocations or amplifications of c-myc. We also show that the frequency of the c-myc coding region mutations in BL is independent of the frequency of mutations in the regulatory region. These results suggest that the mechanisms that induce missense mutations involving the coding region of c-myc may be different from those that lead to mutations in the regulatory regions.

We have analyzed 30 cases of high- and intermediate-grade acquired immunodeficiency syndrome-associated non-Hodgkin's lymphoma (AIDS-NHL) for mutations in the c-myc coding region. In addition, in these same tumors, we have sought the presence of mutations in a regulatory region within the first c-myc intron defined by the binding to a factor that inhibits c-myc transcription (MYC intron factor, or mif). Mutations in the c-myc coding region were present in 10 of 16 small noncleaved cell lymphoma (SNCL), but in only 3 of 14 other histologic subtypes tested (0/3 large non-cleaved cell, 2/8 immunoblastic, and 1/3 anaplastic large cell lymphomas). Nineteen of the AIDS-NHLs analyzed contained a c-myc rearrangement and in 10 of these the c-myc gene was mutated in its coding region. In contrast, we could detect a mutation in the coding region in only 2 of 8 AIDS-NHL without a c-myc rearrangement. Mutations in the mif region were detected in 5 of 16 SNCL. Among AIDS-NHL carrying mutations in the c-myc coding region, only 4 carried mutations in the regulatory region. These results suggest that the mutations in the coding region of the c-myc protein may either be a consequence of the translocations involving c-myc, or may be necessary only in tumors where c-myc is deregulated as a result of a c-myc/lg translocation.

Journal Article A de novo p53 germline mutation affecting codon 151 in a six year old child with multiple tumors Get access Marina I. Gutiérrez, Marina I. Gutiérrez Search for other works by this author on: Oxford Academic PubMed Google Scholar Kishor G. Bhatla, Kishor G. Bhatla * 2Lymphoma Biology Section, Pediatric Branch, National Cancer InstituteBuilding 10, Room 13N240, Betnesda, MD 20892, USA * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Cristina Berreiro, Cristina Berreiro 1Department of Genetics, Hospital de Pediatria ‘Prof. Dr. Juan P.Garrahan’Combate de los Pozos 1881, (1245) Buerios Aires, Argentina Search for other works by this author on: Oxford Academic PubMed Google Scholar Gordon Spangler, Gordon Spangler 2Lymphoma Biology Section, Pediatric Branch, National Cancer InstituteBuilding 10, Room 13N240, Betnesda, MD 20892, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Enrique Schvartzmann, Enrique Schvartzmann Search for other works by this author on: Oxford Academic PubMed Google Scholar Federico Sackmann Muriel, Federico Sackmann Muriel Search for other works by this author on: Oxford Academic PubMed Google Scholar lan T. Magrath lan T. Magrath 2Lymphoma Biology Section, Pediatric Branch, National Cancer InstituteBuilding 10, Room 13N240, Betnesda, MD 20892, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Human Molecular Genetics, Volume 3, Issue 12, December 1994, Pages 2247–2248, https://doi.org/10.1093/hmg/3.12.2247 Published: 01 December 1994 Article history Received: 07 July 1994 Revision received: 08 September 1994 Accepted: 08 September 1994 Published: 01 December 1994

We have screened the entire coding region of c-myc in a panel of Burkitt's lymphomas (BLs) and mouse plasmacytomas (PCTs). Contrary to the belief that c-myc is wild type in these tumours, we found that 65% of 57 BLs and 30% of 10 PCTs tested exhibit at least one amino acid (aa) substitution. These mutations were apparently homozygous in all BL cell lines tested and two tumour biopsies, implying that the mutations often occur before Myc/Ig translocation in BL. In PCTs, only the mutant c-myc allele was expressed indicating a functional homozygosity, but occurrence of mutations after the translocation. Many of the observed mutations are clustered in regions associated with transcriptional activation and apoptosis, and in BLs, they frequently occur at sites of phosphorylation, suggesting that the mutations have a pathogenetic role.

SCL gene rearrangement is the most common molecular lesion (25%) identified so far in T-cell acute lymphoblastic leukemia (T-ALL). Since the frequency of T-ALL appears to be relatively higher in developing countries, we wished to determine as to what fraction of T-ALL from this population harbor SCL rearrangements. We show in this study that although the overall frequency of SCL/SIL rearrangements in T-ALL is similar to the Western countries this is at the expense of increased type A rearrangements. Whether the paucity of type B rearrangements reflects a difference in disease etiology in this part of the world is to be determined.