In the present funding period, we observed that the terminal regions of intracellular Epstein-Barr virus (EBV) genome exhibit marked variation in their restriction-fragment profile in individual, isogenic B-cell lineages cloned directly from three patients. Furthermore, we found that the clonotypic arrays of EBV terminal-region restriction fragments are maintained relatively stably, during in vivo and in vitro proliferation of EBV- transformed cell lineages. The most important implications of these findings are explored here, as follows. First, EBV terminal-fragment analysis can be used to help determine whether EBV infects the progenitor cell of tumor-cell populations, or whether it secondarily infects the tumor population after initial proliferation and dissemination (the """"""""passenger virus"""""""" notion). Here, we propose to analyze the EBV terminal-region fragment in tumor specimens of nasopharyngeal carcinoma, African Burkitt Lymphoma, and AIDS-related B- lymphomas to answer this question. Second, our findings suggest one mechanism of the clonal dominance observed in EBV-related malignancies. The viral gene encoding an important 'early' transmembrane protein (IMP/LYDMA) has been localized by others to a site within about 0.6 kb of the EBV terminal-repeats. The IMP/LYDMA gene product is thought to be important in immune recognition of EBV- infected cells, and possibly in cell transformation. We suggest that the clonal heterogeneity observed in the DNA of intracellular viral terminal regions is associated with cis-acting effects on IMP/LYDMA transcription; evidence provided here indeed indicates clonal variation in IMP/LYDMA transcription. We propose to further analyze the structure and function of intracellular EBV terminal regions. First, the nature and location of clonal heterogeneity of terminal-region DNA will be further analyzed in our cell clones in tumor tissues. Second, quantitative estimates of clonal transcription of IMP/LYDMA will be obtained. Third, clonal variation in the chromatin structure of intracellular EBV terminal regions will be studied. Finally, members of one set of isogenic cell clones will be tested for their individual susceptibility to killing by autologous effector cells, in cytotoxicity assays; and these results will be correlated with the quantitative estimates of clonal transcription of IMP/LYDMA. The proposed studies utilize current findings to discern features of the EBV-terminal region which may be important in clonal dominance and tumorigenesis induced by EBV.
