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J. Biol. Chem., Vol. 278, Issue 32, 29987-29994, August 8, 2003
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¶ **
From the
Department of Medical Genetics and
Microbiology and the Banting and Best Department
of Medical Research, University of Toronto, Toronto, Ontario M5S 1A8, Canada
and the ||Division of Infectious Diseases and
Immunology, Queensland Institute of Medical Research, Brisbane 4029,
Australia
Received for publication, April 15, 2003 , and in revised form, May 22, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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/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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-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 surface (9). This property of EBNA1 has been attributed to the central Gly-Ala repeat, which varies in length in different EBV isolates and is not required for any of the EBNA1 functions measured in tissue culture (10, 11).
EBNA1 has no apparent enzymatic activities and is thought to fulfill its
functions by mediating interactions with specific host cellular proteins.
However, few of these cellular protein interactions have been identified. To
date, only yeast one- and two-hybrid approaches have been used to screen for
EBNA1-interacting proteins; these screens have identified importin-
(also called karyopherin-2 or Rch1), karyopherin-1, p32/TAP, and
EBP2 as EBNA1-binding proteins
(12–17).
Since importin- and karyopherin-1 are known nuclear transport
factors, their interaction with EBNA1 may be important for EBNA1 entry into
the nucleus. p32/TAP (also called gC1q-R) is predominantly a mitochondrial
protein, but has been found to bind to a wide variety of proteins with diverse
functions (Refs. 13 and
18–20
and references therein). Thus, the significance of the interaction of EBNA1
with p32/TAP remains unclear. The interaction of EBNA1 with EBP2 seems to be
important for the segregation function of EBNA1. EBP2 is a component of the
cellular mitotic chromosomes, and EBNA1 appears to attach to EBP2 to partition
EBV plasmids (15,
21,
22). In addition to the
EBNA1-binding proteins identified by screening, studies that have specifically
tested for interactions of EBNA1 with replication protein A and the origin
recognition complex have detected these interactions, suggesting that they
might be important for the replication function of EBNA1
(23,
24).
Since the present set of known protein interactions with EBNA1 is unlikely to account for all of the EBNA1 functions, we have used additional methods to screen for human proteins that specifically recognize EBNA1. Using affinity chromatography and tandem affinity purification (TAP)-tagging approaches, we have identified novel EBNA1-interacting proteins, including the deubiquitinating enzyme USP7 (also known as HAUSP).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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325–376
—EBNA1 (lacking most of the Gly-Ala repeat region) was expressed as
a hexahistidine fusion from a baculovirus. EBNA1 was purified from insect cell
nuclei on a metal chelating column, followed by a heparin-agarose column
(25). 325–376
(lacking amino acids 325–376 in addition to most of the Gly-Ala repeat)
was expressed as a hexahistidine fusion from pET15b in Escherichia
coli and purified as described for EBNA1. EBNA1 Affinity Columns—HeLa S3 cells (10 g; National Cell Culture Center) were lysed in 11 ml of 10 mM HEPES (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), and complete protease inhibitors (Roche Applied Science). 10 ml of 50 mM HEPES (pH 7.5), 1.5 mM MgCl2, 1.26 M NaCl, 0.5 mM DTT, 0.6 mM EDTA, and 75% glycerol was added, and the lysate was homogenized in a Dounce homogenizer. After 30 min on ice, the extract was clarified by centrifugation for3hat 64,000 x g and then dialyzed overnight against 50 mM HEPES (pH 7.5), 20% glycerol, 0.5 mM DTT, 5 mM MgCl2, and 75 mM KCl. Prior to loading onto affinity columns, CaCl2 was added to 4 mM, and lysates were incubated with RNase A and DNase I (1.4 µg/mg of lysate) for 30 min at 25 °C to remove nucleic acid.
Purified EBNA1 or 325–376 was covalently linked to Affi-Gel 10
(Bio-Rad) by incubating 2 ml of protein/ml of Affi-Gel in buffer A (20
mM HEPES (pH 7.5), 10% glycerol, 0.1 mM EDTA, 1
mM DTT, and 1 M NaCl) and blocked as described
previously (26). Coupled resin
was washed with buffer A and equilibrated in buffer A containing 0.1
M NaCl (buffer B) before pouring into 40-µl microcolumns. 400
µl of HeLa lysate (at 14 mg/ml) was applied to the columns. Columns were
washed with 400 µl of buffer B and 160 µl of buffer B containing 1%
Triton X-100 and then sequentially eluted with buffer A and 1% SDS. For
rechromatography assays, the buffer A eluates from three columns were pooled,
dialyzed against buffer A containing 200 mM NaCl, and reapplied to
an EBNA1 affinity column. This column was washed and eluted as described
above. Column eluates were analyzed by SDS-PAGE and silver staining.
Mass Spectrometry—Gel slices containing the protein bands were identified by reduction in DTT, alkylation in iodoacetamide, and then subjection to in-gel trypsin hydrolysis. The peptides were purified and analyzed by MALDI-TOF mass spectrometry using a cyano-4-hydroxycinnamic acid matrix (Sigma) on a Voyager DE-STR instrument (Applied Biosystems) (27). Identification of the proteins using these mass fingerprinting data was carried out using the ProFound software.2
Co-immunoprecipitation from Insect Cells—Baculoviruses
expressing EBNA1GA (with a long Gly-Ala repeat)
(11), EBNA1 or EBNA1 mutants
330–641, 330–619, 452–641, and 41–376 have all
been described previously (28,
29). The baculovirus
expressing EBNA1 with a small Gly-Ala repeat and lacking amino acids
395–450 was constructed by QuikChange mutagenesis (Stratagene) of EBNA1
in pFast-Bac (Invitrogen). Baculovirus was generated according to the
manufacturer's specifications. A baculovirus expressing USP7 with an
N-terminal hexahistidine tag was similarly constructed using USP7
cDNA in pET-3a, kindly provided by Roger Everett
(30). For
co-immunoprecipitation assays, 107 Sf9 cells were infected with
baculovirus expressing USP7 and/or EBNA1 or an EBNA1 mutant. 32 h
post-infection, cells were labeled with 50 µCi of
[35S]methionine for 16 h as described previously
(15). Cells were lysed in 1 ml
of immunoprecipitation buffer (20 mM Tris-HCl (pH 7.5), 200
mM NaCl, 1 mM MgCl2, 10% glycerol, 1% Triton
X-100, and protease inhibitors) on ice for 30 min. After centrifugation, the
supernatant was precleared with protein A-Sepharose and then precipitated with
rabbit anti-EBNA1 polyclonal antibody K67 (kindly provided by Jaap Middeldorp)
and protein A-Sepharose as described previously
(15). Immunoprecipitated
proteins were analyzed by SDS-PAGE, followed by autoradiography of the dried
gels.
TAP-tagging Experiments—The DNAs coding for EBNA1,
395–450, and 325–376 were PCR-amplified from
plasmids containing these EBNA1 mutants and cloned between the XhoI
and NotI sites of pMZI. pMZI expresses proteins with C-terminal TAP
tags (31) in mammalian cells
under the control of an ecdysone-inducible promoter. The pMZI vector and the
pMZI-LacZ construct, which encodes TAP-tagged -galactosidase, will be
described elsewhere.3
293T cells at 60% confluence in 150-mm dishes were cotransfected by calcium
phosphate precipitation with 8 µg of pMZI expressing EBNA1 or an EBNA1
mutant and 8 µg of pVgRxR (Invitrogen), which encodes the ecdysone receptor
heterodimer. The precipitate was removed 15 h post-transfection, and
expression of the EBNA1 proteins was induced by adding medium containing 3
µM Ponasterone A (Invitrogen). The cells were harvested
22–48 h later, and a whole cell extract was prepared from 4 x
107 cells as described by Xiao et al.
(32), except that the extract
was dialyzed against 10 mM HEPES (pH 7.9), 0.1 M
potassium acetate, 0.1 mM EDTA, 0.1 mM DTT, and 10%
glycerol. The extract was mixed with 50 µl of IgG-Sepharose 6 beads
(Amersham Biosciences) for 3 h at 4 °C, and bound TAP-tagged protein was
released by incubation overnight with 30 units of tobacco etch virus (TEV)
protease (Invitrogen) in 10 mM Tris-HCl (pH 8), 100 mM
NaCl, 0.1% Triton X-100, 0.5 mM EDTA, 1 mM DTT, and 10%
glycerol. Eluted protein was further purified on a 40-µl column of
calmodulin-Sepharose 4B. Protein complexes were eluted with 10 mM
Tris-HCl (pH 8), 100 mM NaCl, 2 mM EGTA, 10
mM -mercaptoethanol, 1 mM imidazole, and 0.1%
Triton X-100; concentrated by lyophilization; and analyzed by SDS-PAGE and
silver staining. Protein bands were identified by MALDI-TOF mass
spectrometry.
EBNA1 Functional Assays—The construction of pc3oriPEBNA1 and
pc3oriP, which contain the EBV oriP sequence and express EBNA1 or no
protein, respectively, has been described previously
(15). Plasmid
pc3oriP395–450 was constructed by QuikChange mutagenesis of
pc3oriPEBNA1. Transient replication, plasmid maintenance, and transcriptional
activation assays were performed as described previously
(33). For replication assays,
C33A cells were transfected with 10 µg of pc3oriP plasmids; and 72 h
post-transfection, plasmids were recovered, linearized, digested with
DpnI, and analyzed by Southern blotting. For plasmid maintenance
assays, C33A cells were transfected with 1 µg of plasmid; and after 14 days
of selection in G418, plasmids were linearized, digested with DpnI,
and analyzed by Southern blotting. Plasmid bands were visualized by
autoradiography and quantified by PhosphorImager analysis using ImageQuant
software (Amersham Biosciences). For transactivation assays, C33A cells were
transfected with 5 µg of pc3oriP plasmids and 2 µg of the pFRTKCAT
reporter construct (kindly provided by Bill Sugden). 24 h later, cell lysates
were prepared, and 50 µg of each was assayed for chloramphenicol
acetyltransferase activity using several reaction times as described
previously (34). Reaction
products at each time point were quantified by PhosphorImager analysis and
used to determine the acetylation rate.
Protein Turnover Assays—2 x 106 293T cells
were transfected with 5 µg of pc3oriPEBNA1 or pc3oriP395–450
using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's
recommendations. Cells were grown for 1 week under selection for the plasmid
(400 µg/ml G418) before blocking the cells in 100 µg/ml cycloheximide
(Sigma). Cells were harvested at various times after blocking by resuspending
in 9 M urea and 5 mM Tris-HCl (pH 7.5). Lysates were
clarified by sonication and centrifugation, and 50 µg of each was separated
by SDS-PAGE and Western-blotted using anti-EBNA1 antiserum R4 (raised against
EBNA1-(452–641)). The same lysates were also probed with anti-interferon
regulatory factor 1 (IRF-1) antibody (C20, Santa Cruz Biotechnology).
Immunofluorescence Microscopy—C33A cells expressing EBNA1 were generated by transfecting C33A cells with pc3oriPEBNA1 and growing the cells under selection for the plasmid. The cells were then grown on coverslips to 60% confluence, fixed with formaldehyde (5% (v/v) in phosphate-buffered saline containing 2% sucrose), and permeabilized with acetone/methanol (70:30) at –20 °C. Coverslips were washed with phosphate-buffered saline and blocked for 1 h in 10% bovine serum albumin. The cells were stained with anti-EBNA1 monoclonal antibody OT1x (kindly supplied by Jaap Middeldorp) at a 1:150 dilution and rabbit anti-USP7 antibody r201 (kindly provided by Roger Everett) at a 1:200 dilution, followed by staining with Texas Red-labeled goat anti-mouse (Molecular Probes, Inc.) and fluorescein isothiocyanate-labeled goat anti-rabbit (Invitrogen) secondary antibodies (at 1:200 and 1:30 dilutions, respectively). Cells were then counterstained with 4,6-diamidino-2-phenylindole. The slides were mounted in 5 µl of antifade solution and observed at a magnification of x400 using a Leica DMR microscope and Openlab software. For microscopy of Raji cells, cells were allowed to adhere to coverslips coated with poly-L-lysine before fixing with paraformaldehyde (3% in phosphate-buffered saline), permeabilizing in 1% Triton X-100 and phosphate-buffered saline, and blocking as described above. Staining and microscopy were performed as described for C33A cells, except that rabbit anti-USP7 antiserum r2b2, raised against full-length USP7, was used (at a dilution of 1:200).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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and p32/TAP, were previously identified as EBNA1-interacting
proteins in two-hybrid screens
(12–15).
In addition, previously unknown interactions were detected with USP7,
karyopherin-3, karyopherin-2 (not obvious in
Fig. 1A, but shown in
Figs. 1B and
2), importin-, NAP1
(nucleosome assembly protein-1), template-activating factor (TAF)-I,
TAF-I, CK2 (casein kinase-2), CK2', and pp32.
USP7 is a ubiquitin-specific protease that has been found to interact with the
ICP0 protein of herpes simplex virus type 1
(30). Karyopherin-2 and
karyopherin-3 are nuclear import factors
(35). NAP1 is a histone
chaperone that facilitates the proper spacing of nucleosomes
(36). TAF-I and
TAF-I form the template-activating factor, which can activate
replication and transcription from chromatin templates through histone
interactions (37). In
addition, TAF-I, also known as SET, has been found to regulate
p21Cip1 (38).
CK2 and CK2' are two of the three subunits of the CK2
serine/threonine kinase (39).
pp32 is an acidic nuclear protein that forms part of the INHAT complex
(40). We noted that EBP2,
which was found to bind EBNA1 by other methods
(15), was not among the
proteins retained on the EBNA1 column. However, Western blot analysis of the
soluble HeLa lysate fraction applied to the column indicated that EBP2 was not
present in this extract, but rather had pelleted with the insoluble cellular
material. The EBNA1 affinity column experiment was also performed with a
B-cell lysate and resulted in the same panel of EBNA1-binding proteins as was
isolated from the HeLa lysate (data not shown).
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Since EBNA1 is a highly basic protein (pI 11), we expected that some
cellular proteins containing acidic regions would interact with EBNA1 through
nonspecific ionic interactions. To identify these proteins, we compared the
cellular proteins eluted from the EBNA1 column with those eluted from a column
containing TATA box-binding protein (TBP), which has a pI similar to that
EBNA1. The elution profile from the TBP column shows that the EBNA1-binding
proteins importin-, TAFI-, p32/TAP, and pp32 were also retained
on the TBP column, suggesting that they interact nonspecifically with basic
proteins (Fig. 1A). We
also excised bands from the TBP elution profile that migrated at positions
similar to those of NAP1, importin-, and USP7, but MALDI-TOF analyses
of these bands indicated that NAP1, importin-, and USP7 were not
present in the TBP eluate. We conclude that USP7, NAP1, importin- and
TAF-I interact specifically with EBNA1. Karyopherin- and CK2 bands
were not observed on the TBP column; however, since they are close to the
limit of detection in the EBNA1 profile, we cannot be certain that they are
not present in the TBP profile.
We next tested the relative affinity of the EBNA1-binding proteins for
EBNA1 by rechromatography of EBNA1 column eluates on a second EBNA1 column.
Eluates from three EBNA1 columns were pooled, dialyzed to lower the salt to
100 mM, and then reapplied to another EBNA1 column. When the high
salt elution profiles were compared from the first- and second-pass EBNA1
columns, bands corresponding to USP7, importin-, CK2,
CK2', and p32/TAP were all observed to be enriched upon
rechromatography relative to the other bands
(Fig. 1B), suggesting
that these proteins bind EBNA1 with higher affinity than the other proteins.
Two bands corresponding to NAP1 were identified in this experiment; the lower
band is a proteolytic fragment of the full-length protein.
We have previously shown that EBNA1 amino acids 325–376 are essential
for both the transcriptional activation and partitioning functions of EBNA1,
suggesting that these residues mediate interactions with cellular proteins
involved in these processes
(15,
34). To uncover cellular
proteins that interact with this functionally important region of EBNA1, we
constructed affinity columns using an EBNA1 mutant lacking amino acids
325–376 (325–376) and compared the profile of cellular
proteins retained on this column with that on an EBNA1 column
(Fig. 2). In each of three
experiments, importin- and NAP1 were not detected on the
325–376 column, indicating that residues 325–376 are
important for importin- and NAP1 binding. No other consistent
differences were observed between the EBNA1 and 325–376
profiles.
Mapping of the USP7-interacting Region of EBNA1—The EBNA1-USP7 interaction had not been previously identified and passed all of our criteria for a bona fide interaction, viz. reproducibility, specificity, and strength of binding. To further verify this interaction and to determine whether it is direct or mediated by another human protein, we coexpressed both proteins in insect cells using baculoviruses, metabolically labeled the cells, and performed co-immunoprecipitation experiments using anti-EBNA1 antibody (Fig. 3B). Immunoprecipitates from cell lysates expressing both EBNA1 and USP7 contained labeled bands corresponding to EBNA1 and USP7 (lanes 5 and 14). USP7 also immunoprecipitated with a version of EBNA1 containing a longer Gly-Ala repeat region (EBNA1GA) (lane 13). The USP7 band was not detected in anti-EBNA1 immunoprecipitates from cells expressing either USP7 alone or EBNA1 alone (lanes 8 and 15). Thus, the EBNA1-USP7 interaction does not require other human proteins.
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We used this co-immunoprecipitation assay to determine the region of EBNA1
that interacts with USP7 (Fig.
3B). The series of EBNA1 truncation and deletion mutants
in Fig. 3A were
coexpressed with USP7 in insect cells. In keeping with the affinity column
results, the 325–376 mutant retained the ability to bind USP7
(lane 2). USP7 was also found to interact with EBNA1 mutants lacking
any or all of the N-terminal 395 amino acids, but not with the 452–641
fragment (lane 6), suggesting that residues between amino acids 395
and 452 of EBNA1 mediate the USP7 interaction. In keeping with this
conclusion, an EBNA1 deletion mutant lacking residues 395–450 did not
detectably interact with USP7 in this assay (lane 17).
Identification of Protein Interactions by TAP of EBNA1—We used a TAP-tagging approach to profile cellular proteins that stably interact with EBNA1 in vivo. TAP tagging, which was originally developed for use in yeast, involves expressing the protein of interest fused at the C terminus to a calmodulin-binding peptide, followed by a tobacco etch virus protease cleavage site and a protein A IgG-binding domain (31). The tagged protein is isolated from cell lysates on IgG resin, eluted by tobacco etch virus protease cleavage, further purified on calmodulin resin, and eluted with EGTA. Because the native elution conditions in this procedure enable protein complexes to remain intact throughout the purification, this is a powerful method for profiling in vivo protein interactions. TAP-tagging results also tend to be cleaner than single pull-down experiments since the two-column purification procedure leads to the isolation of only relatively stable protein complexes.
To apply the TAP-tagging approach to EBNA1, we designed a vector expressing
TAP-tagged EBNA1 from an ecdysone-inducible promoter and used it to express
EBNA1 in human 293 cells. TAP-tagged EBNA1 was purified from cell lysates, and
copurifying proteins were separated by SDS-PAGE and identified by MALDI-TOF
mass spectrometry (Fig.
4A). This method identified a subset of cellular proteins
that interacted with EBNA1 in the affinity column approach, viz.
USP7, importin-, CK2 (-, '-, and -subunits),
and p32/TAP. In some experiments, we also identified an interaction with the
protein-arginine methyltransferase PRMT5
(Fig. 4B). PRMT5 was
also found to be retained on EBNA1 affinity columns, but was eluted only with
SDS (data not shown).
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We used the TAP-tagging approach to assess the effects of the
395–450 and 325–376 deletions on cellular protein
interactions with EBNA1 (Fig.
4B). A comparison of the EBNA1 and 395–450
profiles showed that this deletion selectively disrupted the USP7 interaction
without altering interactions with any of the other cellular proteins. This
indicates that the 395–450 mutant is not grossly misfolded and is
suitable for functional studies to assess the significance of the EBNA1-USP7
interaction. Sequence 395–450 likely corresponds to a functional
entity/domain since it is bordered on one side by the DNA-binding domain
(amino acids 452–607) and on the other side by the nuclear localization
signal (amino acids 379–387)
(41). The TAP-tagging results
with 325–376 showed that, like EBNA1, this mutant stably
interacted with USP7, CK2, and p32/TAP, which is consistent with the affinity
column results. A band was also observed at the position of PRMT5, suggesting
that PRMT5 interacts with 325–376. In the experiments in
Fig. 4B, we cannot
comment on interactions with importin- since this band would be
obscured by the bands from the EBNA1 deletion mutants and by the bands
corresponding to partially proteolyzed EBNA1.
Effect of USP7 Interaction on EBNA1 Turnover and
Presentation—EBNA1 is not efficiently processed by the 26 S
proteasome and, as a result, has an extremely long half-life and is not
presented on the cell surface through the major histocompatibility complex
class I pathway. The interaction of EBNA1 with the USP7 ubiquitin protease
suggested that the lack of proteasomal processing of EBNA1 could be due, at
least in part, to the removal of conjugated ubiquitin from EBNA1 by USP7. If
this hypothesis were correct, then the 395–450 mutant, which does
not bind USP7, should remain ubiquitinated and be degraded by the proteasome.
To test this possibility, we compared the turnover of EBNA1 and
395–450 in 293 cells expressing these proteins by following the
amount of each protein remaining at various times after blocking protein
synthesis with cycloheximide (Fig.
5). Little change in the levels of either of the EBNA1 proteins
was observed over the course of the experiment (32 h), indicating that the
395–450 mutation did not significantly alter the stability of
EBNA1 in these cells. Turnover of the unstable IRF-1 protein was observed,
indicating that the cells were blocked by the cycloheximide treatment.
Identical results were obtained when turnover experiments were conducted in
C33A cells and when the 395–450 mutation was made within a
version of EBNA1 containing the full-length Gly-Ala repeat (data not
shown).
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We also tested the presentation of 395–450 mutants (containing
full-length or small Gly-Ala repeats) expressed in B-cells as determined by
lysis by major histocompatibility complex class I-restricted cytotoxic
T-lymphocytes. The results indicated that the 395–450 mutation
did not significantly increase EBNA1 presentation over the levels seen with
the parental EBNA1 proteins (data not shown). Thus, the EBNA1-USP7 interaction
does not appear to be a major determinant in the lack of turnover or
presentation of EBNA1.
Effect of USP7 Binding on the Replication and Transactivation Functions
of EBNA1—We also tested the possibility that USP7 binding may
affect the replication, segregation, and transactivation functions of EBNA1.
Such effects could be due to the ubiquitination state of either EBNA1 or
cellular proteins. To this end, we compared the activities of EBNA1 and
395–450 in plasmid maintenance, replication, and transcriptional
activation assays. Plasmid maintenance assays were performed by transfecting
C33A cells with an oriP plasmid containing the EBV origin of
replication and segregation elements and expressing either EBNA1 or
395–450. After 2 weeks of cell growth, the plasmid levels were
compared by Southern blotting. We observed that 395–450
maintained the plasmids at higher levels (3-fold higher on average) than
EBNA1, whereas oriP plasmids that did not express EBNA1 proteins were
not maintained (Fig.
6A). This effect was not due to abnormally high
expression levels of 395–450, as this protein was expressed at
levels similar to those of wild-type EBNA1 (data not shown). Since stable
plasmid maintenance requires both replication and segregation functions, the
results could reflect an increase in the efficiency of either of these
processes by 395–450. To distinguish between these possibilities,
we measured the replication efficiency of the same plasmids in a transient
replication assay. In this assay, the plasmids were recovered from cells 3
days post-transfection, linearized, and incubated with the
methylation-sensitive enzyme DpnI, which digests the unreplicated
plasmids, leaving replicated plasmids intact. The amount of
DpnI-resistant plasmid was then quantified by Southern blotting. As
shown in Fig. 6B,
oriP plasmids were replicated more efficiently in the presence of
395–450 compared with EBNA1; replication levels calculated from
four experiments were 4-fold higher on average in the presence of
395–450 compared with EBNA1, which could account for the
increased plasmid levels seen in the plasmid maintenance assays. The results
suggest that the EBNA1-USP7 interaction decreases the efficiency with which
EBNA1 activates replication from oriP, but has no obvious effect on
EBNA1 segregation function.
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We also tested the possibility that the USP7 interaction affects the
transcriptional activation function of EBNA1 by comparing the ability of EBNA1
and 395–450 to activate the expression of a chloramphenicol
acetyltransferase reporter gene under the control of the EBV enhancer element
(FR). For these assays, C33A cells were cotransfected with the oriP
plasmids expressing EBNA1 or 395–450 and with pFRTKCAT, and the
level of chloramphenicol acetyltransferase gene expressed was determined by
measuring the acetylation rates for each cell lysate using equal amounts of
protein. Results from four experiments indicated that 395–450 was
slightly less active than EBNA1 in these assays, yielding 65 ± 7% of
the transcriptional activation levels of EBNA1. Thus, the interaction with
USP7 may modestly increase the transcriptional activity of EBNA1, but does not
appear to play a major role in this process.
Cellular Localization and Abundance of USP7 and EBNA1— To better understand the extent of interaction between EBNA1 and USP7 in human cells, we used antibodies raised against USP7 and EBNA1 to determine the localization of these proteins both in an EBV-positive Burkitt's lymphoma cell line (Raji) and in the C33A epithelial cells expressing EBNA1 that were used for the functional assays described above (Fig. 7). In both cases, EBNA1 was found throughout the nucleus, and USP7 was found throughout most of the nucleus, with the exception of the nucleolus. Thus, there is a considerable overlap between EBNA1 and USP7 in interphase cells. One of the C33A cells captured in Fig. 7 was in mitosis, and immunofluorescence staining of this cell showed that USP7 and EBNA1 have very different staining patterns in mitosis; EBNA1 was localized to the mitotic chromosomes, whereas USP7 was found outside of the chromosomes. The localization of EBNA1 and USP7 is therefore consistent with a possible interaction between the two proteins in interphase, but not in mitosis.
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We also compared the amount of USP7 and EBNA1 in Raji cells by Western blot
analyses of Raji whole cell extracts in comparison with known amounts of
purified EBNA1 and USP7. We found that USP7 was very abundant in Raji cells,
with 
130,000 ± 68,000 copies/cell calculated from four separate
experiments. USP7 was also abundant in the C33A cells in which the functional
assays were performed, with a copy number calculated at 50,000 copies/cell.
USP7 was severalfold more abundant than EBNA1 in the Raji cells, which our
experiments indicated was present at 18,000 copies/cell and which has
previously been reported to be present at 37,000 copies/cell
(42). The relative abundance
of EBNA1 and USP7 is consistent with the possibility that a significant
portion of EBNA1 could be bound by USP7 in EBV-infected cells.
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, importin- (also called karyopherin-),
karyopherin-2, and karyopherin-3. The interaction with
importin-, which had been previously detected in yeast two-hybrid
assays (12,
14), was shown to involve
sequence 325–376 of EBNA1. However, the 325–376 mutant,
which is impaired in importin- binding, retains the ability to enter
the nucleus and has wild-type DNA replication activity
(15,
21), suggesting that the
importin- interaction is not solely responsible for nuclear entry or
that a weak interaction with importin- is sufficient for nuclear
transport. Direct interactions with the importin/karyopherin- proteins
may also contribute to nuclear entry. Alternatively, these proteins may serve
as chaperones for EBNA1 since importin- and karyopherin-3 have
both been recently shown to protect exposed basic domains in other proteins
from aggregation (43). Both affinity column and TAP-tagging approaches detected EBNA1 interactions with the p32/TAP protein, which was previously found to bind EBNA1 in yeast two-hybrid assays (13). However, the fact that p32/TAP bound to the TBP column and has been reported to bind numerous basic proteins with no apparent functional connection (18–20) indicates that p32/TAP interacts nonspecifically with basic proteins. This promiscuity makes it unlikely that p32/TAP plays a role in any specific EBNA1 function, but a general role in protein folding or localization remains a possibility. It has been suggested that the interaction with p32/TAP is important for transcriptional activation by EBNA1 (44), but our finding that the EBNA1-p32/TAP interaction was not disrupted by the EBNA1 325–376 deletion, which completely abrogated transcriptional activation, does not support this conclusion.
We also identified previously unknown interactions of EBNA1 with NAP1 and
the related protein TAF-I, both of which have been shown to stimulate
transcription and replication of adenovirus chromatin and to affect nucleosome
positioning through histone interactions
(36,
37,
45). Although these
interactions were detected by EBNA1 affinity chromatography, they were not
detected in TAP-tagging experiments and were not enriched upon multiple rounds
of EBNA1 affinity chromatography, suggesting that the interactions are
relatively weak or transient. The EBNA1 interaction with NAP1, but not with
TAF-I, was found to be disrupted by the 325–376 deletion, which also
abrogated the transcriptional activity of EBNA1. This suggests that NAP1 might
mediate transcriptional activation by EBNA1 through chromatin remodeling.
EBNA1 interactions with TAF-I or TAF-I did not correlate with
transcriptional activity, but could be important for other EBNA1 functions
such as activation of DNA replication and cell immortalization. TAF-I
and TAF-I are used in multiple ways to affect cellular gene expression
and cell cycle progression. TAF-I and TAF-I have been shown to be
components of the INHAT complex, which inhibits histone acetylation
(40); and TAF-I, also
know as SET, has been shown to be associated with myeloid leukemia
(46) and to interact with
p21Cip1 to potentially regulate cell cycle progression
(38). Although our data
suggest that the EBNA1 interaction with TAF-I may occur through
nonspecific charge interactions, it is interesting that the interaction with
TAF-I, which can function independently of TAF-I, appears to be
specific. It is also worth noting that the pp32 protein, which was found to
interact with EBNA1 through nonspecific charge interactions, is functionally
similar to TAF-I in that it also interacts with histones and is a component of
the INHAT complex (40).
The remaining three new EBNA1-interacting proteins identified in this study (CK2, PRMT5, and USP7) are involved in post-translational modifications. Thus, interactions with these proteins could reflect the fact that EBNA1 is modified by these proteins and/or that EBNA1 affects the modification of cellular proteins by binding these proteins. The EBNA1 interaction with CK2 was consistently observed by both the affinity chromatography and TAP-tagging methods. CK2 is a serine/threonine kinase that has many cellular targets and is implicated in several cellular pathways, including cell cycle progression, malignant transformation, and regulation of apoptosis (39). Thus, the interaction of EBNA1 with CK2 may affect any of these pathways. The EBNA1-CK2 interaction may also reflect the regulation of EBNA1 function by CK2 phosphorylation, as EBNA1 is known to be phosphorylated at serine residues (25) and contains three putative CK2 sites.
PRMT5 (also known as JBP1) is a protein-arginine methyltransferase and is the human homolog of the fission yeast Skb1 (47). Increasing evidence points to the importance of arginine methylation in regulating a variety of protein functions (48). Methylation most often occurs at RGG sequences, a motif that occurs multiple times in EBNA1 in the functionally important region 325–376 as well as in region 33–56 (49). Thus, the EBNA1-PRMT5 interaction detected in TAP-tagging experiments could indicate that EBNA1 is methylated by PRMT5 and/or that EBNA1 influences cellular processes regulated by PRMT5. To date, PRMT5 has been implicated in signal transduction, the assembly of small nuclear ribonucleoprotein complexes, and cell proliferation (47, 50, 51).
The interaction of EBNA1 with USP7 was identified by both affinity chromatography and TAP-tagging approaches and further verified by co-immunoprecipitation of the coexpressed proteins in insect cells. This nuclear ubiquitin-specific protease 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 herpes simplex virus type 1 lytic infectious cycle (30). ICP0 is a ubiquitin-protein isopeptide ligase that promiscuously activates gene expression and induces the destruction of specific cellular proteins (52). Although the significance of the USP7-ICP0 interaction is not fully understood, the ability of ICP0 to bind USP7 correlates with its ability to activate gene expression, suggesting a role for USP7 in this process (53). We have now shown that a protein (EBNA1) from another herpesvirus (Epstein-Barr virus) also binds USP7, suggesting that USP7 may be a common target of herpesviruses.
The interaction of EBNA1 with USP7 suggested that EBNA1 might be deubiquitinated by USP7 and that this removal of conjugated ubiquitin from EBNA1 might be part of the mechanism by which EBNA1 avoids proteasomal degradation. This possibility was supported by the finding that EBNA1 that had been ubiquitinated in vitro was deubiquitinated by purified USP7 (data not shown). However, an EBNA1 mutant defective in USP7 binding exhibited the long half-life and lack of major histocompatibility complex class I presentation typical of wild-type EBNA1, indicating that USP7 binding is not responsible for preventing the turnover and presentation of EBNA1. Several studies have shown that the Gly-Ala repeat of EBNA1 is important in preventing the turnover and presentation of EBNA1, although the mechanism by which this sequence inhibits proteasomal targeting is not clear (9–11). Our results are consistent with the findings that the Gly-Ala repeat is the dominant sequence preventing proteasomal degradation of EBNA1.
We also explored the possibility that disruption of USP7 binding to EBNA1
by the 395–450 mutation affects the functions of EBNA1.
395–450 was found to replicate oriP-containing plasmids
4-fold more efficiently than wild-type EBNA1, resulting in higher maintenance
levels of these plasmids. The results are consistent with a role for USP7 in
regulating EBNA1 replication function, and we envisage two models of how USP7
might influence this process. The first model assumes that the effects are due
to deubiquitination of EBNA1 by USP7; and therefore, the degree of EBNA1
ubiquitination affects its ability to function in replication. In this case,
ubiquitination would stimulate EBNA1 replication activity, presumably by
affecting specific protein interactions. This could be due to
monoubiquitination rather than polyubiquitination since monoubiquitination is
emerging as a post-translational modification that can regulate protein
function without affecting turnover
(54,
55). The second model assumes
that the effects are due to deubiquitination of cellular proteins by USP7. In
this model, EBNA1 would bring USP7 to oriP, where it could
deubiquitinate one or more cellular proteins that affect the activation of
replication. This could mean either that ubiquitination makes a cellular
factor more active for DNA replication or that ubiquitination inactivates (or
targets for proteasomal destruction) a negative regulator of DNA
replication.
Although our studies have initially focused on how the EBNA1-USP7 interaction affects EBNA1 functions, it is also likely that sequestering of USP7 by EBNA1 affects cellular functions that are normally regulated by USP7. USP7 was recently shown to bind and deubiquitinate p53, resulting in p53 stabilization and p53-dependent growth arrest and apoptosis (56). Conversely, a catalytically inactive USP7 point mutant caused increased p53 ubiquitination and destabilization. If EBNA1 efficiently sequesters or inactivates USP7, then we would expect EBNA1 to destabilize p53, thereby promoting cell cycle progression and inhibiting apoptosis. Such effects may be important for host cell immortalization by EBV as well as in the development of EBV-associated tumors. The possibility that EBNA1 directly contributes to these processes is supported by the fact that some EBV tumors express only EBNA1 and by the ability of EBNA1 to induce B-cell neoplasia in transgenic mice (8). It will be interesting to determine how the interaction of EBNA1 with USP7 and the other cellular partners identified here contributes to EBV-induced immortalization and tumorigenesis.
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FOOTNOTES |
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¶ Present address: Lab. d'Enzymologie et Biochimie Structurales, CNRS, Bat.
34, 1 Avenue de la Terrasse, 91198 Gif sur Yvette, France.
** Canadian Institutes of Health Research Scientist. To whom correspondence should be addressed: Dept. of Medical Genetics and Microbiology, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-946-3501; Fax: 416-978-6885; E-mail: lori.frappier{at}utoronto.ca.
1 The abbreviations used are: EBV, Epstein-Barr virus; EBNA1, Epstein-Barr
nuclear antigen-1; TAP, tandem affinity purification; DTT, dithiothreitol;
MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TAF,
template-activating factor; TBP, TATA box-binding protein.
2 Available at 129.85.19.192
[EC]
/profound_bin/WebProFound.exe.
3 M. Zeghouf, J. Li, G. Butland, A. Borkowska, V. Canadien, D. Richards, B.
Beattie, A. Emili, and J. Greenblatt, submitted for publication.
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