Intranuclear distribution of Epstein-Barr virus-encoded nuclear antigens EBNA-1, -2, -3 and -5

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Abstract
Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) express at least seven virally encoded proteins. Their functional role, and their relationships to each other and to normal nuclear constituents are virtually unknown. As the first step towards a topographical study, the intranuclear distribution of EBV-encoded nuclear antigens EBNA-1, -2, -3 and -5 (abbreviated El, E2 etc.) was examined in EBV-transformed LCLs by immunofluorescence and digital image analysis of fluorescence patterns. E1-E3 showed a finely granular distribution. The E2 patterns were virtually identical when comparing indirect staining using an E2-specific mouse monoclonal antibody with anticomplement immunofluorescence using a human antibody, rendered monospecific to E2 by absorption. The E1/E2 patterns showed 32% overlap and the E2/E3 10% overlap in the high overlap category (66.7-100%), while the E2/E2 comparison with two reagents showed 61 % overlap in this category. This suggests that E2 and E3 largely appear in different nuclear structures, whereas El appears to be randomly distributed with regard to E2. The E5 pattern was radically different from that of El, E2 and E3. The anti-E5 mouse monoclonal antibody detected 4-10 huge, globular, sharply circumscribed dots, located in dispersed chromatin areas, while the distribution of El, E2 and E3 showed no obvious relationship to chromatin distribution. The methods described here allow a more refined topographical analysis of the EBNA protein family, mostly in relation to each other, in relation to other nuclear proteins, and with respect to specialized functional domains in interphase chromatin. The topology of the EBNA proteins relative to other nuclear components may provide important clues about the role of growth transformation-associated proteins in cellular immortalization and viral latency.