Protein Profiling with Epstein-Barr Nuclear Antigen-1 Reveals an Interaction with the Herpesvirus-associated Ubiquitin-specific Protease HAUSP/USP7

Abstract

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...

A RING Finger Ubiquitin Ligase Is Protected from Autocatalyzed Ubiquitination and Degradation by Binding to Ubiquitin-specific Protease USP7

“…It is more likely to be a proofreading enzyme that regulates the stability of specific target proteins by antagonizing their polyubiquitination. Indeed, it has been shown that USP7 specifically deubiquitinates p53 in vitro and in vivo (34) and has a similar effect on the Epstein-Barr virus regulatory protein EBNA-1 (32). In that USP7 binds to both p53 and EBNA-1, it may be that its binding partners are protected from ubiquitination by USP7 activity.…”

“…1, D and E). Higher amounts of USP7 (on the order of 1 g in similar assays instead of the 5 ng used here) have, however, been shown to cleave a similar model isopeptidelinked substrate (32), and therefore the protein does not lack the potential for this activity.…”

“…has been shown to bind to and cleave ubiquitinated forms of both p53 and Epstein-Barr virus regulatory protein EBNA-1 (32,34); although in the case of p53, the K m value was estimated to be greater than 50 M (33). As a control for the intrinsic activity of our preparations of USP7, we generated ubiquitinated p53 in reactions using ICP0 as the E3 ubiquitin ligase (14).…”

“…Vitro-Previous reports have demonstrated that USP7 has ubiquitin-specific protease activity in vitro, both on model ␣-peptide-linked ubiquitin substrates and on isopeptide-linked p53 ubiquitin conjugates (32)(33)(34). We expressed and purified a His-tagged version of wild type USP7 (Fig.…”

Epstein–Barr virus oncogenesis and the ubiquitin–proteasome system

“…Resistance to proteolysis may be required for the capacity of EBNA-1 to act as a transcriptional regulator either alone or together with interacting cellular proteins. Indeed, using both affinity chromatography and TAP-tagging approaches, Holowaty et al (2003b) have recently shown that EBNA-1 interacts with the isopeptidase USP7, also known as herpesvirus-associated ubiquitin-specific protease, HAUSP. This nuclear enzyme was first identified by virtue of its interaction with the ICP0 protein of herpes simplex virus type 1, which is required for efficient initiation of the HSV-1 lytic cycle (Everett et al, 1997).…”