Nuclear compartmentalization of viral and cellular proteins involved in gamma-herpes virus (EBV and HHV-8) mediated cellular transformation

Sammanfattning: The aim of the work described in this thesis has been to characterize the nuclear compartmentalization of cellular and viral proteins involved in EBV or HHV-8 mediated cellular transformation. We have studied the EBV encoded EBNA-5 and its association with the PML nuclear bodies (NBs) and nuclear inclusions. We showed that in EBV infected cells the EBNA-5 protein accumulated in NBs. The NBs accumulates a number of cellular proteins, including the PML protein (promyelocytic leukemia protein) and are targeted by various viral proteins. In acute promyelocytic leukemia the NBs are disrupted. Several recent findings indicate that the NBs are involved in proteasomal protein degradation. We wanted to study a possible involvement of the NBs and its component EBNA-5 in the proteasomal protein degradation. We found that inhibition of the proteasomal-protein degradation machinery induced a coordinated change in localization and the translocation of both endogenous and transfected EBNA-5 along with Hsp70 and mutant p53 to the nucleoli. This may reflect the involvement of EBNA-5 in the regulation of intracellular protein trafficking, possibly associated with the proteasome-mediated degradation. The suggested association of PML protein and NBs with proteasome-mediated protein degradation prompted us to analyze the intracellular distribution of proteasomes, different NB components and non-NB associated proteins in the presence of a proteasome inhibitor. Inhibition of the proteasomes in different cell lines resulted in a radical redistribution of the NB-associated proteins PML, Sp100, SUMO-1 and EBNA-5 along with the proteasomes themselves. The accumulation of NBassociated proteins and proteasomes in the nucleoli of MG132-treated cells indicates that these proteins may target the nucleoli under normal conditions and that the nucleolus may have a function in the regulation of proteasomal protein degradation. EBNA-5 interacts with the p14ARF protein, a regulator of the p53 pathway. We have shown that in the presence of EBNA-5, the p14ARF localized to the nucleoli but also in extra-nucleolar inclusions. In addition, the inclusions contained p53 and HDM2, and were surrounded by NBs and proteasomes. p14ARF was shown to induce the relocation of HDM2 and p53 to these extranucleolar sites. Accumulation of PML and proteasomes at these sites suggest that the components of the nuclear inclusions are targeted for proteasome-mediated degradation. We have also studied the influence of HHV-8 encoded latency associated nuclear antigen (LANA-1) on heterochromatin. LANA-1 is the major latency associated nuclear protein of HHV-8. HHV-8 is a newly identified human herpes virus that is the likely causative agent of Kaposis sarcoma. The virus is also present in body cavity lymphomas and in multicentric Castleman's disease. We have shown that LANA1 accumulates in heterochromatinassociated nuclear bodies and stays on the mitotic chromosomes during cell division. We showed that LANA-1 induces the expression of its cellular partner RING3, a human homologue of the Drosophila female sterile homeotic (fsh) gene. LANA-1 relocates RING3 from the euchromatin to the heterochromatin borders. High levels of exogenously expressed LANA-1 caused a reorganization of heterochromatin in both human and mouse cells. Our recent results demonstrate that it is the internal acidic repeat of LANA-1 that is responsible for the re-organization of heterochromatin. We could also show that LANA-1 change the organization of heterochromatin-associated proteins such as histone H3 (trimethyl K9) and MeCP2 in parallel with the re-organization of heterochromatin. We suggest that LANA may create a local euchromatic microenvironment around the viral episomes that are anchored to the heterochromatin.