P32/TAP, a Cellular Protein That Interacts with EBNA-1 of Epstein–Barr Virus

Abstract

The Epstein-Barr virus (EBV) EBNA-1 protein has a central role in the maintenance of a latent EBV infection and is the only virus-encoded protein expressed in all EBV-associated tumors. EBNA-1 is required for replication of the episomal form of the latent viral genome and transactivates the latency C and LMP-1 promoters. The mechanisms by which EBNA-1 performs these functions are not known. Here we describe the cloning, expression, and characterization of a cellular protein, P32/TAP, which strongly interacts with EBNA-1. We show that P32/TAP is expressed at high levels in Raji cells and is synthesized as a proprotein of 282 amino acids (aa) that is posttranslationally processed by a two-step cleavage process to yield a mature protein of 209 aa. It has been previously reported that P32/TAP is expressed on the cell surface. Our transient expression assays detected full-length P32/TAP (1-282 aa) in the cytoplasm while mature P32/TAP protein localized to the nucleus. Three lines of evidence support P32/TAP interaction with EBNA-1. First, in the yeast two-hybrid system we mapped two interactive N-terminal regions of EBNA-1, aa 40-60 and aa 325-376, each of which contains arginine-glycine repeats. These regions interact with the C-terminal half of P32/TAP. Second, the full-length cytoplasmic P32/TAP protein can translocate nuclear EBNA-1 into the cytoplasm. Third, P32/TAP co-immunoprecipitated with EBNA-1. We have confirmed that a Gal4 fusion protein containing the C-terminal region of P32/TAP (aa 244-282) transactivates expression from a reporter containing upstream Gal4-binding sites. Deletion of the P32/TAP interactive regions of EBNA-1 severely diminished EBNA-1 transactivation of FRTKCAT in transient expression assays. Our data suggest that interaction with P32/TAP may contribute to EBNA-1-mediated transactivation.

Maintenance of Epstein-Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

“…In one study, deletion of EBNA-1 amino acids 325-376 had almost no effect on initial DNA replication but nearly eradicated episome persistence (31). The basic domains are also implicated in the looping between EBNA-1 bound to FR and DS, in linking among EBNA-1 molecules and in interactions with cellular proteins, one of which, EBP2, is associated with mitotic chromosomes (27)(28)(29)(30)(31)40).…”

“…Notably, the entire EBNA-1 C terminus, which includes the DNA-binding and dimerization domains, and NLS, cannot bind chromosomes. Although further dissection of the EBNA-1 basic domains could more precisely define the components critical for chromosome association and plasmid maintenance, these domains are also implicated in transcriptional activation, DNA looping and linking, and homo-and heterotypic protein-protein interactions (11,(26)(27)(28)(29)(30)(31). Because specific mutations would therefore likely have pleiotropic effects, we chose an alternative approach; to replace the EBNA-1 chromosome associating domains with cellular proteins that were similar to EBNA-1 in the pattern and salt sensitivity of their association with chromosomes.…”

Epstein–Barr virus nuclear antigen 1 activates transcription from episomal but not integrated DNA and does not alter lymphocyte growth

“…However, although EBNA1 is unlikely to interact with cognate cell DNA to affect cell gene expression or growth, we cannot dismiss the possibility that EBNA1 may interact with cell proteins and affect cells through pathways that may not have been detectable in the experiments described here. EBNA1 N-terminal residues have been implicated in interaction with the cellular proteins p32 and EBP2, and interaction with EBP2 is important in episome persistence (48,49). Also, EBNA1 amino acids 91-321 are a glycine-alanine repeat domain that can inhibit proteasome-mediated degradation of EBNA1 (20).…”

Modelling the structure of full-length Epstein–Barr virus nuclear antigen 1

“…EBNA1 associates with multiple host proteins including ubiquitin-specific protease 7 (USP7), casein kinase 2 (CK2) (36,37), EBP2 and others (26,(37)(38)(39)(40)(41). The core interaction sites of EBNA1 with USP7 and CK2 have been determined and map to the central region of the protein (37,42).…”