ABSTRACT
The Epstein-Barr virus (EBV) EBNA-LP protein is important for EBV-mediated B-cell immortalization and is a potent gene-specific coactivator of the viral transcriptional activator, EBNA2. The mechanism(s) by which EBNA-LP functions as a coactivator remains an important question in the biology of EBV-induced B-cell immortalization. In this study, we found that EBNA-LP interacts with the promyelocytic leukemia nuclear body (PML NB)-associated protein Sp100 and displaces Sp100 and heterochromatin protein 1alpha (HP1alpha) from PML NBs. Interaction between EBNA-LP and Sp100 was mediated through conserved region 3 in EBNA-LP and the PML NB targeting domain in Sp100. Overexpression of Sp100 lacking the N-terminal PML NB targeting domain, but not a mutant form of Sp100 lacking the HP1alpha interaction domain, was sufficient to coactivate EBNA2 in a gene-specific manner independent of EBNA-LP. These findings suggest that Sp100 is a major mediator of EBNA-LP coactivation. These studies indicate that modulation of PML NB-associated proteins may be important for establishment of latent viral infections, and also identify a convenient model system to investigate the functions of Sp100.
Subject(s)
Antigens, Nuclear/metabolism , Autoantigens/metabolism , B-Lymphocytes/virology , Cell Nucleus/metabolism , Epstein-Barr Virus Nuclear Antigens/metabolism , Gene Expression Regulation, Viral , Nuclear Proteins/metabolism , Viral Proteins/metabolism , Antigens, Nuclear/analysis , Antigens, Nuclear/genetics , Autoantigens/analysis , Autoantigens/genetics , Cell Nucleus/chemistry , Cells, Cultured , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/metabolism , Epstein-Barr Virus Nuclear Antigens/genetics , Humans , Nuclear Proteins/analysis , Nuclear Proteins/genetics , Protein Interaction Mapping , Protein Structure, Tertiary , Sequence Deletion , Transcription, Genetic , Up-Regulation/genetics , Viral Proteins/analysisABSTRACT
The tumor suppressor protein PML and oncoprotein MDM2 have opposing effects on p53. PML stimulates p53 activity by recruiting it to nuclear foci termed PML nuclear bodies. In contrast, MDM2 inhibits p53 by promoting its degradation. To date, neither a physical nor functional relationship between PML and MDM2 has been described. In this study, we report an in vivo and in vitro interaction between PML and MDM2 which is independent of p53. Two separate regions of PML are recognized which can interact with MDM2. The C-terminal half of PML, encoded by residues 300-633, can interact with the central region of MDM2 which includes the MDM2 acidic domain. In addition, PML amino acids 1-200, which encode the RING-finger and most of the B box zinc binding motifs, can interact with the C-terminal, RING-finger containing region of MDM2. Interestingly, PML mutants in which sumoylation at lysine 160 was inhibited displayed an increased association with MDM2, suggesting that sumoylation at this site may be a determinant of PML-MDM2 binding. Coexpression with MDM2 caused a redistribution of PML from the nucleus to the cytoplasm, and this required the PML N terminus and the MDM2 RING-finger domain. These results suggest that interaction between the PML N terminus and MDM2 C terminus can promote PML nuclear exclusion. Wild-type MDM2 inhibited the ability of PML to stimulate the transcriptional activity of a GAL4-CBP fusion protein. This inhibition required the central, acidic region of MDM2, but did not require the MDM2 C terminus. Taken together, these studies demonstrate that MDM2 and PML can interact through at least two separate protein regions, and that these interactions can have specific effects on the activity and/or localization of PML.