Four peptides were synthesized on the basis of amino acid sequences deduced from a highly spliced transcript encoded by the Bam W, Y, and H fragments of the Epstein-Barr virus (EBV) genome [Bodescot, M., Chambraud, J. B., Farrell, P. J. & Perricaudet, M. (1984) EMBOJ. 3, 1913-1917. Rabbit antisera against three of the four peptides identified a nuclear polypeptide that varied between 22 and 70 kDa in molecular size. Four of 20 EBV-positive human sera contained antibodies against this polypeptide.Since this is the fifth EBV-determined nuclear antigen (FBNA) discovered in grQwth-transformed cells, it is designated EBNA5. The antigen was detected in virus nonproducer lines (<0.01 % EBV early antigen expression) and is thus not dependent on the viral cycle. It was differentially expressed depending on the orign of the lines. Al 10 lymphoblastoid cell lines tested expressed EBNA5, but it could not be detected in 10 of 11 EBV-carrying Burkitt lymphoma lines. Infection of tonsillar lymphocytes with the B95-8 strain of EBV induced six EBNA5-specific polypeptides that varied between 41 and 70 kDa in molecular size with regular increments of 6 kDa. This may be due to the fact that the EBNA5 coding sequence includes the Bam W internal repeat. Parallel infection of the EBVnegative Burkitt lymphoma line Ramos with the same viral substrain did not induce detectable levels of EBNA5, nor was this antigen present in permanently EBV-converted Ramos sublines. These findings imply that the expression of the viral genome varies among B cells having different phenotypes.

Somatic cell hybrids were generated between an MCF-virus-induced 15-trisomic T-cell lymphoma of AKR origin with a proviral insertion near the c-myc locus, and normal diploid fibroblasts or lymphocytes of CBAT6T6 origin. Three lymphoma/fibroblast fusions were performed. Six independently-derived clones from 2 fusions were tested for tumorigenicity. Three of the 6 clones were weakly malignant (take incidence 20% below), and 3 were strongly malignant (take incidence over 80%). All 3 lymphoma/lymphocyte hybrids and 6 derived clones were strongly malignant. All hybrids contained a nearly complete chromosomal complement of both parental cells. This was confirmed at the molecular level by determining the ratio of germ-line (G) vs. rearranged (R) myc-carrying Eco RI fragments that showed the expected 1.9-2.7:1 proportion. Malignant segregants selected from the weakly malignant lymphoma/fibroblast hybrids by in vivo inoculation showed changed 15-chromosome ratios. Four out of the 6 clones showed amplification of the lymphoma-derived 15-chromosome that carries the R-myc fragment and a concomitant decrease in the average number of the G-myc-carrying chromosomes. This was deduced from the fact that the G:R ratio was between 2 and 3:1 in the in vitro hybrids but became inverted (1:2-3) in the tumors. Two tumors showed no amplification of R-myc. G-myc was decreased. One of these tumors showed a change in the G:R ratio from 2.5:1.0 to 1.2:1.0, while the other was essentially unchanged (1.9:1.0 in the in vitro clone and 2.2:1.0 in the derived tumor). These findings support the notion that both the amplification of the lymphoma-derived 15-chromosome with the retrovirally rearranged c-myc carrying fragment and/or the loss of the G-myc-carrying 15-chr can contribute to the tumorigenic potential of the hybrids.

Six independently maintained sublines of FLEB 14, a fetal-liver-derived Epstein-Barr virus-transformed pro-B cell line that has not yet rearranged its immunoglobulin genes, were examined after in vitro propagation during 19-36 months. Two lines showed no immunoglobulin heavy chain gene rearrangement, whereas one allele was rearranged with breakpoints inside the switch region of the mu chain gene in the remaining four. These rearrangements had been generated by the translocation of different chromosome fragments to the immunoglobulin heavy chain gene cluster-carrying 14q32 band in each of the four lines. Previously, a similar rearrangement was found in a fifth subline concurrently with a reciprocal 6;14 translocation. The transposed pieces have been derived from chromosomes 16 and 18 in two of the more recently rearranged lines. Their origins could not be determined in the remaining two lines, but they were different from each other and the other three 14q+ markers. The 14q+ marker-carrying variant has replaced its diploid progenitor suggesting that the translocation has conveyed some in vitro growth advantage on its carrier. This was also supported by the duplication of the 14q+ marker and the loss of its normal chromosome 14 homologue in one subline during serial culturing. The vulnerability of the switch region of the mu chain gene to illegitimate recombination at the pro-B stage and the possible relevance of this finding for the origin of the Burkitt lymphoma-associated 8;14 (immunoglobulin heavy chain gene cluster/MYC) translocation is discussed.

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Somatic cell hybrids were generated between YACUT, a doubly drug-resistant subline of YAC-1 (a Moloney-virus-induced T-cell lymphoma of strain A/Sn origin with 2 proviral insertions near the pvt-1 locus) and normal diploid fibroblasts of CBAT6T6 origin. Three independent fusions were performed. Three uncloned hybrid cultures and 9 independently-derived clones were tested for tumorigenicity by the inoculation of graded cell numbers into syngeneic hosts. One of 3 uncloned hybrid cultures and 3 of 9 clones were weakly tumorigenic (take incidence 0%), and 1 of 3 uncloned hybrid cultures and 6 clones were highly tumorigenic (take incidence greater than 80%). One weakly tumorigenic hybrid and 3 weakly tumorigenic clones carried 3 copies of the tumor-derived chromosome 15 and 2 copies of the normal fibroblast-derived t(14;15) chromosomes. In contrast, 2 highly malignant hybrid clones lost one copy of the normal-fibroblast-derived t(14;15), but contained increased numbers (3.44-4.44) of the tumor-derived chromosome 15. Four tumorigenic segregants selected from the weakly tumorigenic fibroblast hybrids by in vivo inoculation showed the same cytogenetic change as the highly tumorigenic hybrid clones, in that the ratio of the normal:tumor-derived chromosomes 15 changed from 1.18-1.55 to 4.11-5.71. Tumorigenicity was thus associated with a modified balance between the tumor vs. the normal-parent-derived 15-chromosomes. Instead of the usual 3:2 ratio, the tumor-derived 15-chromosomes increased disproportionately, whereas the relative number of the normal-parent-derived 15-chromosome decreased, as a rule. These results suggest that amplification of the lymphoma-derived chromosome 15 favors tumorigenicity, but that this effect is counteracted by some influence emanating from the normal-parent-derived homologous chromosome.