USP7/HAUSP is a key regulator of p53 and Mdm2 and is targeted by the Epstein-Barr nuclear antigen 1 (EBNA1) protein of Epstein-Barr virus (EBV). We have determined the crystal structure of the p53 binding domain of USP7 alone and bound to an EBNA1 peptide. This domain is an eight-stranded beta sandwich similar to the TRAF-C domains of TNF-receptor associated factors, although the mode of peptide binding differs significantly from previously observed TRAF-peptide interactions in the sequence (DPGEGPS) and the conformation of the bound peptide. NMR chemical shift analyses of USP7 bound by EBNA1 and p53 indicated that p53 binds the same pocket as EBNA1 but makes less extensive contacts with USP7. Functional studies indicated that EBNA1 binding to USP7 can protect cells from apoptotic challenge by lowering p53 levels. The data provide a structural and conceptual framework for understanding how EBNA1 might contribute to the survival of Epstein-Barr virus-infected cells.

The Epstein-Barr nuclear antigen-1 (EBNA1) protein of Epstein-Barr virus is important for the replication, segregation, and transcriptional activation of latent Epstein-Barr virus genomes; has been implicated in host cell immortalization; and avoids proteasomal processing and cell-surface presentation. To gain insight into how EBNA1 fulfills these functions, we have profiled cellular protein interactions with EBNA1 using EBNA1 affinity chromatography and tandem affinity purification (TAP) of EBNA1 complexes from human cells (TAPtagging). We discovered several new specific cellular protein interactions with EBNA1, including interactions with HAUSP/USP7, NAP1, template-activating factor-I␤/SET, CK2, and PRMT5, all of which play important cell regulatory roles. The ubiquitin-specific protease USP7 is a known target of herpes simplex virus, and the USP7-binding region of EBNA1 was mapped to amino acids 395-450. A mutation in EBNA1 that selectively disrupted binding to USP7 was found to cause a 4-fold increase in EBNA1 replication activity but had no effect on EBNA1 turnover and cell-surface presentation. The results suggest that USP7 can regulate the replication function of EBNA1 and that EBNA1 may influence cellular events by sequestering key regulatory proteins.Epstein-Barr virus (EBV) 1 is a ubiquitous human ␥-herpesvirus that persists for the life of the host. As part of its latent infectious cycle, EBV immortalizes the host cell and, in doing so, predisposes the cell to malignant transformation. As a result, EBV is associated with several types of cancer. EBV genomes are maintained in latently infected replicating cells as circular DNA episomes that replicate once per cell cycle and segregate stably during cell division (reviewed in Refs. 1 and 2). Epstein-Barr nuclear antigen-1 (EBNA1) is the only viral protein required to maintain the EBV genomes in proliferating cells, which it does by binding to recognition sites in the FR (family of repeats) and DS (dyad symmetry) elements of the latent origin of DNA replication, oriP (3, 4). EBNA1 binding to the DS element is necessary to initiate DNA replication from this element (5). EBNA1 binding to the FR element is important for the partitioning of the EBV episomes during cell division and also activates the expression of other viral latency genes (6). In addition to its functions at oriP, EBNA1 has been shown to repress its own transcription (7) and to promote the development of B-cell lymphomas in transgenic mice, suggesting a direct role for EBNA1 in cell transformation (8).While fulfilling all of its functions, EBNA1 avoids detection by host cytotoxic T-lymphocytes. This ability to hide from the immune system is biologically important, as it enables the persistence of latently infected cells that express EBNA1 in the absence of other EBV antigens. The failure of EBNA1 to elicit a cytotoxic T-lymphocyte response is due to lack of proteasomal processing, which prevents the presentation of EBNA1 by major histocompatibility complex class I molecules on the cell surfa...

USP7 or HAUSP is a ubiquitin-specific protease in human cells that regulates the turnover of p53 and is bound by at least two viral proteins, the ICP0 protein of herpes simplex type 1 and the EBNA1 protein of EpsteinBarr virus. We have overexpressed and purified USP7 and shown that the purified protein is monomeric and is active for cleaving both a linear ubiquitin substrate and conjugated ubiquitin on EBNA1. Using partial proteolysis of USP7 coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we showed that USP7 comprises four structural domains; an N-terminal domain known to bind p53, a catalytic domain, and two C-terminal domains. By passing a mixture of USP7 domains over EBNA1 and ICP0 affinity columns, we showed that the N-terminal p53 binding domain was also responsible for the EBNA1 interaction, while the ICP0 binding domain mapped to a C-terminal domain between amino acids 599 -801. Tryptophan fluorescence assays showed that an EBNA1 peptide mapping to residues 395-450 was sufficient to bind the USP7 N-terminal domain and did so with a dissociation constant of 0.9 -2 M, whereas p53 peptides spanning the USP7-binding region gave dissociation constants of 9 -17 M in the same assay. In keeping with these relative affinities, gel filtration analyses of the complexes showed that the EBNA1 peptide efficiently competed with the p53 peptide for USP7 binding, suggesting that EBNA1 could affect p53 function in vivo by competing for USP7.

The EBNA1 protein of Epstein-Barr virus (EBV) plays essential roles in enabling the replication and persistence of EBV genomes in latently infected cells and activating EBV latent gene expression, in all cases by binding to specific recognition sites in the latent origin of replication, oriP. Here we show that EBNA1 binding to its recognition sites in vitro is greatly stimulated by binding to the cellular deubiquitylating enzyme, USP7, and that USP7 can form a ternary complex with DNA-bound EBNA1. Consistent with the in vitro effects, the assembly of EBNA1 on oriP elements in human cells was decreased by USP7 silencing, whereas assembly of an EBNA1 mutant defective in USP7 binding was unaffected. USP7 affinity column profiling identified a complex between USP7 and human GMP synthetase (GMPS), which was shown to stimulate the ability of USP7 to cleave monoubiquitin from histone H2B in vitro. Accordingly, silencing of USP7 in human cells resulted in a consistent increase in the level of monoubquitylated H2B. The USP7-GMPS complex formed a quaternary complex with DNA-bound EBNA1 in vitro and, in EBV infected cells, was preferentially detected at the oriP functional element, FR, along with EBNA1. Down-regulation of USP7 reduced the level of GMPS at the FR, increased the level of monoubiquitylated H2B in this region of the origin and decreased the ability of EBNA1, but not an EBNA1 USP7-binding mutant, to activate transcription from the FR. The results indicate that USP7 can stimulate EBNA1-DNA interactions and that EBNA1 can alter histone modification at oriP through recruitment of USP7.

USP7 (also called HAUSP) is a de-ubiquitinating enzyme recently identified as a key regulator of the p53-mdm2 pathway, which stabilizes both p53 and mdm2. We have discovered that the Epstein-Barr nuclear antigen 1 protein of Epstein-Barr virus binds with high affinity to USP7 and disrupts the USP7-p53 interaction. The results have important implications for the role of Epstein-Barr nuclear antigen 1 in the cellular immortalization that is typical of an Epstein-Barr virus latent infection.