We have previously shown that Epstein-Barr virus (EBV)-encoded EBNA-5 is localized to PML bodies (PODs) in EBV-immortalized lymphoblastoid cell lines (LCLs). Here we have extended our study of the subnuclear localization of EBNA-5 and found a strict co-localization with PML in LCLs and in BL lines with an immunoblastic, LCL-like phenotype. Moreover, GFP-EBNA-5 accumulated in PML bodies upon transfection into LCLs. In contrast, transfection of cell lines of non-immunoblastic origin with an EBNA-5 expression construct showed preferential localization of the protein to the nucleoplasm. Since PML is involved in proteasome-dependent protein degradation, we investigated the total levels and sub-cellular localization of EBNA-5 upon inhibition of proteasome activity. We found that a proteasome inhibitor, MG132, induced the translocation of both endogenous and transfected EBNA-5 to the nucleoli in every cell line tested. The total EBNA-5 protein levels were not affected by the proteasomal block. EBNA-5 forms complexes with heat shock protein Hsp70. The proteasome inhibitor induced a rise in total levels of Hsp70 and dramatically changed its homogeneous nuclear and cytoplasmic distribution into nucleolar and cytoplasmic. This effect was EBNA-5-independent. The nucleolar localization of Hsp70 was enhanced by the presence of EBNA-5, however. EBNA-5 also enhanced the nucleolar translocation of a mutant p53 in a colon cancer line, SW480, treated with MG132. The coordinated changes in EBNA-5 and Hsp70 localization and the effect of EBNA-5 on mutant p53 distribution upon MG132 treatment might reflect the involvement of EBNA-5 in the regulation of intracellular protein trafficking associated with the proteasome-mediated degradation.