Epstein-Barr Nuclear Antigen 1 Contributes to Nasopharyngeal Carcinoma through Disruption of PML Nuclear Bodies

Latent Epstein-Barr virus (EBV) infection is strongly associated with several cancers, including nasopharyngeal carcinoma (NPC), a tumor that is endemic in several parts of the world. We have investigated the molecular basis for how EBV latent infection promotes the development of NPC. We show that the viral EBNA1 protein, previously known to be required to maintain the EBV episomes, also causes the disruption of the cellular PML (promyelocytic leukemia) nuclear bodies (or ND10s). This disruption occurs both in the context of a native latent infection and when exogenously expressed in EBV-negative NPC cells and involves loss of the PML proteins. We also show that EBNA1 is partially localized to PML nuclear bodies in NPC cells and interacts with a specific PML isoform. PML disruption by EBNA1 requires binding to the cellular ubiquitin specific protease, USP7 or HAUSP, but is independent of p53. We further observed that p53 activation, DNA repair and apoptosis, all of which depend on PML nuclear bodies, were impaired by EBNA1 expression and that cells expressing EBNA1 were more likely to survive after induction of DNA damage. The results point to an important role for EBNA1 in the development of NPC, in which EBNA1-mediated disruption of PML nuclear bodies promotes the survival of cells with DNA damage

EBNA1-Mediated Recruitment of a Histone H2B Deubiquitylating Complex to the Epstein-Barr Virus Latent Origin of DNA Replication

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

The Herpesvirus Associated Ubiquitin Specific Protease, USP7, Is a Negative Regulator of PML Proteins and PML Nuclear Bodies

The PML tumor suppressor is the founding component of the multiprotein nuclear structures known as PML nuclear bodies (PML-NBs), which control several cellular functions including apoptosis and antiviral effects. The ubiquitin specific protease USP7 (also called HAUSP) is known to associate with PML-NBs and to be a tight binding partner of two herpesvirus proteins that disrupt PML NBs. Here we investigated whether USP7 itself regulates PML-NBs. Silencing of USP7 was found to increase the number of PML-NBs, to increase the levels of PML protein and to inhibit PML polyubiquitylation in nasopharyngeal carcinoma cells. This effect of USP7 was independent of p53 as PML loss was observed in p53-null cells. PML-NBs disruption was induced by USP7 overexpression independently of its catalytic activity and was induced by either of the protein interaction domains of USP7, each of which localized to PML-NBs. USP7 also disrupted NBs formed from some single PML isoforms, most notably isoforms I and IV. CK2α and RNF4, which are known regulators of PML, were dispensable for USP7-associated PML-NB disruption. The results are consistent with a novel model of PML regulation where a deubiquitylase disrupts PML-NBs through recruitment of another cellular protein(s) to PML NBs, independently of its catalytic activity

Functions of Ubiquitin Specific Protease 7 (USP7) in Epstein-Barr Virus Infection and Associated Cancers

The Epstein-Barr virus (EBV) infects over 90% of the human population and is associated with several human malignancies. The EBNA1 protein of EBV binds recognition sites in the latent origin of replication (oriP) and is important for the replication and segregation of EBV genomes in latently-infected cells. EBNA1 is also directly implicated in malignant transformation and immortalization of the host cell. EBNA1 does not have any known enzymatic activity and it employs cellular proteins to mediate its functions. One such protein is the ubiquitin specific protease, USP7, which is a key regulator of the p53 tumor suppressor. The aim of this thesis was to functionally characterize the interaction between EBNA1 and USP7. Here I show that USP7 promotes the DNA-binding activity of EBNA1 and is recruited along with an accessory protein, GMPS, to the oriP. The USP7-GMPS complex can deubiquitinate histone H2B and may enable epigenetic regulation of latent viral infection. Additionally, I present evidence for a direct role of EBNA1 in EBV-mediated carcinogenesis. EBNA1 prevents stabilization of p53 by USP7 and abrogates p53 activation by disrupting promyelocytic leukemia nuclear bodies (PML-NBs) that acetylate p53. This interferes with p53-activated gene expression and inhibits apoptosis. EBNA1-expressing cells also have impaired ability to repair DNA, but survive as well as or better than control cells. Thus EBNA1 creates a cellular environment conducive to transformation and immortalization. These studies have also allowed me to learn more about and expand on the known functions of USP7. I provide biochemical evidence suggesting that a P/A/ExxS motif is a preferred sequence for binding the USP7 N-terminal domain. Furthermore, I show USP7 is a negative regulator of PML proteins and PML-NBs and promotes p53 DNA-binding activity. Surprisingly, neither function required the deubiquitinase activity of USP7.Ph

USP7/HAUSP promotes the sequence-specific DNA binding activity of p53.

The p53 tumor suppressor invokes cellular responses to stressful stimuli by coordinating distinct gene expression programs. This function relies heavily on the ability of p53 to function as a transcription factor by binding promoters of target genes in a sequence specific manner. The DNA binding activity of the core domain of p53 is subject to regulation via post-translational modifications of the C-terminal region. Here we show that the ubiquitin specific protease, USP7 or HAUSP, known to stabilize p53, also regulates the sequence-specific DNA binding mediated by the core domain of p53 in vitro. This regulation is contingent upon interaction between USP7 and the C-terminal regulatory region of p53. However, our data suggest that this effect is not mediated through the N-terminal domain of USP7 previously shown to bind p53, but rather involves the USP7 C-terminal domain and is independent of the deubiquitylation activity of USP7. Consistent with our in vitro observations, we found that overexpression of catalytically inactive USP7 in cells promotes p53 binding to its target sequences and p21 expression, without increasing the levels of p53 protein. We also found that the USP7 C-terminal domain was sufficient for p21 induction. Our results suggest a novel mode of regulation of p53 function by USP7, which is independent of USP7 deubiquitylating activity

USP7 promotes p53 DNA-binding in vivo.

<p>H1299 cells were either transfected with an empty plasmid (Vector) or transfected with a p53-expressing plasmid alone or in combination with constructs expressing either WT myc-tagged USP7 (+USP7 WT) or myc-tagged C223S (+C223S). p53 occupancy of various promoters in transfected cells was measured by chromatin immunoprecipitation using a p53 antibody and Q-PCR of the target sequences indicated. GAPDH was used a negative control region for Q-PCR. Results were normalized to p53 levels determined for each sample by Western blotting (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013040#pone-0013040-g004" target="_blank">Figure 4B</a> for an example) to adjust for any small variations in p53 levels.</p

Effects of USP7 NTD and CTD on p53 DNA binding.

<p>(A) Schematic representation of the USP7 proteins used in this study showing the N-terminal (NTD) or TRAF domain, central catalytic (CAT) and C-terminal (CTD) domains. The position of the C223S point mutation that inactivates the catalytic domain is also shown. (B–C) EMSAs showing titration of latent p53_82–393, in the presence or absence of 20 µM USP7-NTD (B) or 20 µM USP7-CTD (C). Quantification of the discreet shifted bands are shown in the graphs, with USP7 in black and BSA in grey.</p

Catalytically inactive USP7 stimulates p53 function.

<p>(A) U2OS cells were transfected with an empty vector or vector expressing the myc-tagged C223S mutant of USP7 and treated with etoposide for 0, 1, 2 or 4 hours as indicated. Equal amounts of cell lysates were analyzed for protein expression by western blotting using the indicated antibodies where actin is the loading control. (B) H1299 cells were either transfected with an empty plasmid (Vector) or transfected with a p53-expressing plasmid alone or in combination with constructs expressing myc-tagged USP7 and USP7 mutants as indicated. 24 hours post-transfection, cells were lysed and protein levels were measured by Western blotting using the antibodies indicated. The higher p53 band in lane 4 corresponds to monoubiquitylated p53 which becomes apparent due to the dominant-negative effects of C223S <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013040#pone.0013040-Li1" target="_blank">[24]</a>. (C) H1299 cells were transfected with plasmids expressing p53 and myc-tagged USP7-CTD and a luciferase reporter construct in the combinations indicated. 48 hours post-transfection, cells were lysed and luciferase activity was quantified. The results are shown relative to the p53 plus reporter sample for three independent experiments.</p