Identification of EBNA binding cellular proteins, using yeast two-hybrid system

Abstract: Epstein-Barr virus (EBV) is a common herpesvirus that establishes life-long persistence in the human host through the elaborate regulation of different latency types. Latent EBV infection of resting B cells converts them into transformed cells that can develop into tumors in immune-compromised hosts. As such, EBV infection is a unique, well-defined in vitro system for malignant transformation. The latency- associated EBV proteins are the key factors for virus-induced cell transformation. To fully understand the molecular mechanisms that lead to virusinduced transformation, the cellular targets of the transforming viral proteins have to be identified. During the work summarized in this thesis, the following EBV encoded nuclear antigen (EBNA) binding proteins were identified: epsilon-subunit of the human chaperonin TCP- 1 complex; XAP2, the minor subunit of the arylhydrocarbon receptor (AhR) complex; a novel human uridine kinase/uracil phosphoribosyltransferase (for EBNA-3); and p14ARF (for EBNA-5). Epsilon-subunit of the human chaperonin TCP-1 complex is part of a huge protein complex that helps to fold the newly synthesized polypeptides. We mapped the interacting region to the apical domain of the epsilon-subunit the most likely site for recognition of newly translated polypeptides. We concluded that nascent EBNA-3 might receive help for its folding from the TCP-1 complex. Another protein that binds to EBNA-3 is XAP-2, which also interacts with the transformation associated X-protein encoded by the hepatitis B virus. We showed that EBNA-3 induces translocation of the cytoplasmic XAP-2 to the nucleus. We also found a previously unknown EBNA-3 binding protein, which was designated F538. We have shown that the predominantly cytoplasmic F538 relocated to the nucleus in the presence of EBNA-3, where these two proteins showed high levels of co-localization. A natural splice variant with the deleted C-terminus of F538 did not translocate to the nucleus. A SWISSModel 3D structure of F538 was constructed and compared with other known proteins. This raised the possibility that F538 is a novel human uridine kinase/uracil phosphoribosyltransferase (UK/UPRT). We suggested that EBNA-3 by direct protein- protein interaction induced the nuclear accumulation of this enzyme that is most likely part of the ribonucleotide salvage pathway. Increased intra-nuclear levels of UK/UPRT might contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation. We found that EBNA-5 binds to p14ARF, one of the upstream regulators of the p53 pathway. We showed that EBNA-5 prolonged the survival of the p14ARFtransfected cells. We observed the accumulation of the p14ARF in extra-nuclear inclusions where it co-localized with p53, HDM2 and Hsp70. Formation of the p14ARF inclusions induced the translocation of PML bodies and 20S proteasome subunits. Co-expression of p14ARF and EBNA-5 led to the complete relocation of EBNA-5 into the p14ARF inclusions. We suggested that EBNA-5 might play a role in regulating the degradation of p14ARF-p53-HDM2 complexes. In conclusion, using the yeast two-hybrid system we found new cellular targets for EBV-encoded transformation associated latent proteins. Some of these target molecules participate in signal transduction (Xap-2); growth associated metabolic pathways (F538); cell cycle regulation and protein degradation (p14ARF).