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

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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).</p><h3>List of scientific papers</h3><p>I. Kashuba E, Pokrovskaja K, Klein G, Szekely L (1999). Epstein-Barr virus-encoded nuclear protein EBNA-3 interacts with the epsilon-subunit of the T-complex protein 1 chaperonin complex. J Hum Virol. 2(1): 33-7. <br><a href="https://pubmed.ncbi.nlm.nih.gov/10200597">https://pubmed.ncbi.nlm.nih.gov/10200597</a><br><br></p><p>II. Kashuba E, Kashuba V, Pokrovskaja K, Klein G, Szekely L (2000). Epstein-Barr virus encoded nuclear protein EBNA-3 binds XAP-2, a protein associated with Hepatitis B virus X antigen. Oncogene. 19(14): 1801-6. <br><a href="https://pubmed.ncbi.nlm.nih.gov/10777214">https://pubmed.ncbi.nlm.nih.gov/10777214</a><br><br></p><p>III. Pokrovskaja K, Mattsson K, Kashuba E, Klein G, Szekely L (2001). Proteasome inhibitor induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5. J Gen Virol. 82(Pt 2): 345-58. <br><a href="https://pubmed.ncbi.nlm.nih.gov/11161273">https://pubmed.ncbi.nlm.nih.gov/11161273</a><br><br></p><p>IV. Kashuba E, Kashuba V, Sandalova T, Klein G, Szekely L (2002). Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human uridine kinase/uracil phosphoribosyltransferase. BMC Cell Biol. 3(1): 23. <br><a href="https://pubmed.ncbi.nlm.nih.gov/12199906">https://pubmed.ncbi.nlm.nih.gov/12199906</a><br><br></p><p>V. Kashuba E, Matsson K, Pokrovskaja K, Kiss C, Protopopova M, Ehlin-Henriksson B, Klein G, Szekely L (2002). EBV-encoded EBNA-5 associates with p14ARF in extranucleolar inclusions and prolongs the survival of p14ARF-expressing cells. [Submitted]</p><p>VI. Kashuba E, Matsson K, Klein G, Szekely L (2002). Accumulation of p14ARF protein in extranucleolar inclusions induces the re-distribution of PML bodies and proteasomes. [Submitted]</p>