Impact of the stronger binding affinity of EBNA2 259-435 compared to that of

EMSAs proved the higher binding affinity of the EBNA2-truncation EBNA2259-435 to RBP-Jκ compared to that of NotchRamANK. Depending on the oligonucleotide used for complex formation with RBP-Jκ the affinity is 20 to 50 times higher than that of NotchRamANK.

The sequence of the oligonucleotide bound to RBP-Jκ has a strong influence on the binding affinity of ternary complex partners of RBP-Jκ. It is possible that the alteration of certain bases causes a conformational change in RBP-Jκ sufficient to influence its binding affinity to other proteins.

EBNA2 is necessary to maintain proliferation in EREB2.5 cells, a conditionally immortalized lymphoblastoid cell line, where EBNA2 is fused to the hormone-binding domain of the estrogen receptor, rendering its activity dependent on estrogen (Kempkes 1995). Independently it was found that Notch-IC, introduced in different ways, was not able to fully substitute for EBNA2 in these cells, when EBNA2 expression was abolished by estrogen depletion (Hofelmayr et al., 2001). Another group found, however, that expression of very high levels of Notch-IC, obtained by repeated selection for EREB2.5 cells expressing higher than average levels of Notch-IC could finally rescue EREB2.5 cells after inactivation of EBNA2 by estrogen depletion (Gordadze et al., 2001). It was found that Notch-IC could not increase the expression of LMP1, another EBV protein necessary

Taking into account that different RBP-Jκ binding oligonucleotides result in differences in affinity between the respective ternary complex partners NotchRamANK and EBNA2259-435 a possible explanation can be proposed. LMP1 expression is not activated by Notch-IC, but by EBNA2. The RBP-Jκ binding sequence within this promoter corresponds to one used in the present study (Figure 3.39), which showed that EBNA2259-435 binds more strongly to RBP-Jκ bound to this sequence than NotchRamANK. The transactivation activity of EBNA2 and the lack of transactivation when Notch replaces EBNA2 could partly be explained by the dependence of the binding affinity of RBP-Jκ for the complex partners on the oligonucleotide in the complex, but probably even more by the generally stronger binding of EBNA2. Binding of two weakly interacting molecules can be enhanced if 1) other proteins are mediating the interaction as scaffolds or 2) the concentration of one or both interaction partners is increased so that the binding equilibrium is driven towards formation of the complex. In the case of Notch-IC overexpressed at unusually high levels exactly this could have been the case. The excess supply of Notch-IC shifts the equilibrium, especially as RBP-Jκ is anyway generally expressed at moderate levels.

Both Notch-IC and EBNA2 were shown to act similarly on several promoters, but also differences have been found, which, apart from the different binding affinity to RBP-Jκ, could arise from their slightly different binding sites on RBP-Jκ resulting in slightly different conformational changes in RBP-Jκ. Furthermore, the most intriguing difference between EBNA2 and Notch, apart from the PEST sequence labeling Notch for rapid breakdown by the ubiquitin-proteasome pathway, lies in the extracellular domain of Notch, which enables a tight control of the expression of all Notch1 target genes. EBNA2 does not possess any such control element.

In general, a signaling protein is involved not only in a single isolated pathway but in several pathways with different feedback loops, creating a complicated and still largely unknown network, which provides the appropriate reaction of cells upon different sets of stimuli. In the present case, only one pathway is examined, excluding the possible influence of others, which could also involve Notch and/or EBNA2.

A set of additional interacting proteins at the transcription initiation level is already known for both EBNA2 and Notch-IC. Components of the basal transcription machinery interact with both proteins (TFIIH, TBP) as well as histone acetyltransferases (EBNA2: p300/CBP and PCAF; Notch-IC: p300/CBP, PCAF and GCN5) and proteins binding to RBP-Jκ

An important question concerns the origin of the generally higher binding affinity of EBNA2259-435 compared to NotchRamANK and the impact of this higher affinity on the infection process of Epstein-Barr virus.

Up to 95% of individuals may be infected by EBV depending on the origin of human populations. The high affinity of viral proteins for other cellular proteins may well contribute to high levels of infection as the displacement of the usual interaction partners puts the infected cells into a latency phase rather than resulting in cell destruction upon virus production.

During latency infected resting B-cells do not express EBV proteins. Even in many of the malignant diseases caused by EBV especially EBNA2 is not expressed. It is produced, though, in tumours developed by immunocompromised patients, such as those infected with HIV.

The applicability for only a small percentage of patients whose tumours are caused by EBV together with the findings in the present work, that EBNA2 has a much stronger affinity for RBP-Jκ than Notch-IC raise questions regarding the chances of success of a therapeutic attack at this stage of the viral cycle. Since EBNA2 and Notch-IC bind to at least adjacent sites on RBP-Jκ and compete for the binding a drug will most likely expel Notch-IC rather than EBNA2 from RBP-Jκ.