Down-regulation of proteolytic complexes following EBV activation in BL cells
In Burkitt's lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin-proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the alpha/beta subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6' subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.
Down-regulation of proteolytic complexes following EBV activation
in BL cells
, Alessandra De Leo
, Riccardo Gavioli
, Livia Di Renzo
, Elena Mattia
Department of Public Health Sciences, University ‘‘La Sapienza’’, P. le A. Moro 5, 00185 Rome, Italy
Department of Experimental Medicine and Pathology, University ‘‘La Sapienza’’, V. le Regina Elena 324, 00161 Rome, Italy
Department of Biochemistry and Molecular Biology, University of Ferrara, Via Borsari 46, 44100 Ferrara, Italy
Received 17 November 2006
Available online 4 December 2006
In Burkitt’s lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin–proteasome system to promote
episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In
the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induc-
tion of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially
reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer
time after induction. Protein levels of the a/b subunits of the 20S proteasome and TPPII concentration decreased to diﬀerent extents
after EBV activation, whereas the ubiquitin binding S6’ subunit of the 19S regulatory complex increased three to fourfold along with
the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems
at the onset of EBV lytic infection.
2006 Elsevier Inc. All rights reserved.
Keywords: Epstein Barr virus; Lytic cycle; Proteasome; TPPII
The Epstein Barr virus (EBV) is an ubiquitous human
herpes virus that replicates in infected cells establishing
either a latent or a lytic infection.
Three types of latent programs have been characterized
depending on the diﬀerential expression of a restricted
number of viral gene s (reviewed in [1,2]). These include six
nuclear antigens (EBNA1, EBNA2, EBNA-3A, -3B, -3C,
and EBNA-LP), and three latent membrane proteins
(LMP1, LMP2A, and LMP2B). Entry into the viral lytic
cycle is triggered by expression of either of the two EBV
immediate-early (IE) genes, BZLF1 and BRLF1 which func-
tion as transcriptional activators and initiate the sequential
activation of viral lytic gene expression culminating in
release of infectious viral particles and usually death of the
host cell (reviewed in ).
EBV is strongly associated with both B-cell and epithe-
lial cell malignancies .
All EBV-associated malignancies have a predo minantly
latent pattern of viral gene expression. However, many
EBV-associated tumors are poor targets for the cellular
immune response suggesting that imm une evasion strate-
gies might favor tumor development.
The proteasome–ubiquitin proteolytic machinery is
involved in a wide variety of cellular processes that
include cell cycle progression, apoptosis, signal transduc-
tion, transcriptional regulation, receptor internalization,
and the generation of antigenic peptides that trigger
MHC-class I-restricted T cell responses . The protea-
some consists of a 20S cylindrical proteol ytic core
that binds 19S regulatory complexes at its ends. The
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved.
Corresponding author. Fax: +39 0649914641.
E-mail address: Elena.Mattia@uniromal.it (E. Mattia).
Biochemical and Biophysical Research Communications 352 (2007) 947–952
association of these elements form s the 26S proteasome,
which is involved in the targeted degradation of ubiqui-
tin-conjugated proteins . The 20S core is made out of
28 subunits classiﬁed as a and b type. The three active
proteolytic sites are contained in the b subunits, b1, b2,
and b5 which exhibit post-acidic (caspase-like), trypsin-
like, and chymotrypsin-like activity, respect ively .
Following exposure of cells to IFN-c, these subunits are
substituted with new components. The modiﬁed protea-
some, known as immunoproteasome , is characterized
by a reduced post-acidic activity and by an increased
cleavage after hydrophobic and basic residues. Further-
more, it has been shown to promote a more eﬃcient gen-
eration of T lymphocyte epitope s [9,1 0].
EBV, like other tumor viruses [11,12], promotes the
maintenance of latency and eludes the host immune
response by interacting with the ubiquitin–proteasome
system. Viral products shown to be involved in this
interaction are the latent proteins LMP2A, EBNA1,
and LMP1 . Ubiquitination of LMP2A and of the
signaling molecules Lyn and Syk aﬀects BCR-mediated
activation of the EBV lytic cycle [14–16]. The long
repetitive sequence of Gly and Ala (GAr) residues on
the amino terminal half of EBNA1 impedes processing
of the protein by the proteasome, aﬀecting thereby the
generation of peptides that can associate with MHC
class I molecules . LMP1 binding to TRAF-2 and
-3 prevents ubiquitin-mediated degradation of these pro-
teins contributing to uncontrolled B cell proliferation
. Moreover, proteasome degradation of LMP1 could
generate peptide fragments of strong immunosuppressive
Modulation of the ubiquitin–proteasome system is also
concomitant to deregulated expression of cellular onco-
genes. In fact, it has been shown that in EBV-positive
Burkitt’s lympho ma (BL) cells over-expressing c-Myc,
the enzymatic activity of the proteasomes is largely
reduced. Nevertheless, proteolysis proceeds unperturbed
since de-ubiquitinating enzymes and the serine protease
tripeptidylpeptidase II (TPPII) are highly expressed in
these cells .
TPPII is a cytosolic serine protease that removes tripep-
tides from free N-terminus of oligopeptides [21,22]. The
core subunit of about 138 kDa forms large oligomeric com-
plexes that are detected in the cytoplasm or associated with
the plasma membrane of many cell types . It may act
downstream of the proteasomes further digesting partially
degraded proteins ; it may participate in antigen pro-
cessing  and compensate or replace some proteasomal
At present, it is unknown whether EBV interacts with
the cellular proteolytic systems also during the lytic phase
of infection. To address this question, in this study we have
analyzed the activities and the expression of the ubiquitin–
proteasome and of the TPPII proteolytic systems in two
EBV-positive BL cell lines, after induction of the EBV lytic
Materials and method s
Cell culture and EBV lytic cycle induction. EBV positive BL lines Raji
and Akata were cultured in RPMI 1640 medium containing 5% fetal calf
serum (FCS) and antibiotics, in a 5% CO
atmosphere and maintained at a
cell density of 3.5 · 10
For induction, Raji cells were treated with TGF-b, sodium butyrate
and P(BU)2 as described  and Akata cells were treated with goat anti-
human IgG .
Enzymatic assays. At diﬀerent times, after the addition of EBV lytic
cycle inducers, approximately 5 · 10
cells were centrifuged for 5 min at
400g, washed three times with ice-cold PBS and resuspended in lysis
buﬀer (50 mM Tris–HCl, pH 7.4, 5 mM MgCl
, 250 mM sucrose, 1 mM
DTT, and 2 mM ATP). Samples were frozen at 80 C and thawed
three times before being centrifuged for 10 min at 15,000g. Enzymatic
activities were determined in the supernatant fractions using the ﬂuor-
ogenic substrates (100 lM) Succ-LLVY-AMC (chymotryptic-like), Z-
ARR-AMC (trypsin-like), Z-LLE-AMC (caspase-like) and AAF-AMC
(TPPII) (all from Calbiochem) in a buﬀer containing 50 mM Tris–HCl,
pH 7.4, 5 mM MgCl
, and 1 mM DTT. Equal fractions of the super-
natants were pre-incubated with the proteasome inhibitor MG132 or
lactacystine (both at 10 lM), or the TPPII inhibitor AAF-CMK at
30 lM (all inhibitors were obtained from Biomol) for 30 min at 37 C
before addition of the substrates. The reactions were blocked by
addition of cold ethanol and the ﬂuorescence determined by ﬂuorimeter
(Kontron SFM 25) with an excitation wavelength of 380 nm and an
emission wavelength of 460 nm. The enzymatic activities were adjusted
for the protein concentration of the sample, determined by a modiﬁed
Lowry assay (RC DC protein Assay, Bio-Rad).
Western blotting. Raji cells were incubated with TGF-b, sodium
butyrate, and P(BU)2, as described . At the indicated times, samples
cells) were collected by a short centrifugation (5 min at 400g), washed
with PBS and lysed in sample buﬀer (2% SDS, 5% b-mercaptoethanol,
0.001% bromophenol blue, 0.06 M Tris, pH 6.8, and 10% glycerol). To
detect ubiquitinated proteins, cells were lysed in RIPA buﬀer (50 mM
Tris–HCl, pH 7.4, 1% NP40, 0.1% SDS, and 0.5% sodium deossycolate)
containing 2 mM N-ethylmaleimide (Sigma) to prevent deubiquitination.
Cell lysates were mixed with 4· concentrated sample buﬀer and sonicated
three times for 5 s at 10 V. Equal amounts of proteins (70 lg), determined
by RC-DC Protein Assay (Bio-Rad) were separated by 10% SDS–PAGE
and electroblotted to PVDF membrane (Amersham Pharmacia Biotech).
Primary antibodies used: anti 20S proteasome a/b subunits (Biomol); anti-
S6’ proteasome subunit (Aﬃnity BioReagents); anti ubiquitin (Sigma) and
anti-TPPII (IMMUNSYSTEM). Speciﬁc signals detected by chemolumi-
nescence (ECL, Amersham Pharmacia Biotech) were quantiﬁed by den-
sitometric analysis using the ImageJ free-share software (http://
Results and discussion
In EBV-positive Burkitt’s lymphoma cells, reactivation
of the EBV lytic cycle can be obtained eﬃciently with dif-
Raji cells represent an useful model for study of EBV
activation since a large proportion (>60%) of the popula-
tion becomes positive for the early antigens after incuba-
tion with TGF-b, sodium butyrate and P(BU)2  .
However, late antigens expression is blocked and cells do
not lyse . On the other hand, induction of the lytic cycle
in Akata cells by anti-IgG antibodies triggers the full cas-
cade of lytic gene expression with the producti on of viral
particles, closely reﬂecting physiological mechanisms which
control the transition from latency to viral reactivation
948 G. Matusali et al. / Biochemical and Biophysical Research Communications 352 (2007) 947–952
In order to study the activity of the cellular proteolytic
complexes following induction of the EBV lytic cycle, Raji
and Akata cells untreated or incubated with lytic cycle
inducers were harvested and lysed. Chymotrypsin-like,
trypsin-like and caspase-like (post-acidic) activities of the
proteasome, and the activity of the TPPII were assayed
using the ﬂuorogenic substrates Suc-LLVY-AMC, Z-
ARR-AMC, Z-LLE-AMC, and with AAF-AMC, respec-
tively. In order to distinguish between speciﬁc and unspe-
ciﬁc enzymatic activities, before addition of the
substrates, equal amounts of the supernata nts were incu-
bated with MG132 or lactacystine, inhibitors of proteaso-
mal activity, or the AAF-CMK inhibitor of TPPII
activity. The unspeciﬁc activity, i.e. that measur ed in the
presence of each inhibitor, did not vary signiﬁcantly in
the samples taken at diﬀerent times of EBV lytic cycle
induction (data not shown). Therefore, it was considered
to be proteasome- or TPPII-speciﬁc only the activity sup-
pressible by their respective inhibitors.
The three enzymatic activities of the proteasome were
determined in Raji cells at 24, 48 and 72 h after addition
of EBV lytic cycle inducers and the mean values are report-
ed in the bar-graph of Fig. 1A as percentage of the activity
of control cells. It appears that chymotrypsin-like and cas-
pase-like activity of the Raji proteasome were reduced in
contrast to the trypsin-like activity which remained sub-
stantially stable during induction of the EBV lytic cycle.
In particular, 72 h after induction of EBV lytic cycle, chy-
motrypsin-like and caspase-like activities were reduced to
about 20 and 50% of the control, respectively. Measure-
ments carried out in the presence of the proteasome inhib-
itor lactacystine were substantially similar to those
obtained with MG132 (data not shown).
The results observed with Raji cells were conﬁ rmed in
Akata cells in which the EBV lytic cycle was induced by
anti-IgG. In this cell line, about 30% of the cell population
became positive for EBV-early antigens when tested by
immunoﬂuorescence (data not shown). As the lytic cycle
goes to completion in these cells, we limited our studies
to the ﬁrst 24 h of treatment when the majority of cells
(>90%) were still viable. As illustrated in Fig. 1B, chymo-
trypsin-like and caspase-like activities of the proteasome
were signiﬁcan tly reduced upon induction of the EBV lytic
cycle. Evaluation of the inhibitor suppressible activities
indicates that after 24 h of incubation with anti IgG, chy-
motrypsin- and caspase-like activities of the proteasomes
were reduced by 85% and 90%, respectively whereas tryp-
sin-like activity was about 20% lower than that measured
in untreated Akata cells.
For both Raji and Akata cells, a comparison of the
hydrolysis of the ﬂuorogenic substrates that monitor chy-
motrypsin- trypsin- and caspase-like activities of the pro-
teasome suggested that chymotrypsin-like activity was the
main enzymatic function of this proteolytic complex, con-
ﬁrming what has been previously observed .
Fig. 2 illustrates the results for TPPII activity in Raji
and Akata cells treated with EBV inducers in the absence
or in the presence of the TPPII inhibitor AAF-CMK.
TPPII speciﬁc activity of Raji cells (Fig. 2A), was reduced
by 90% during the ﬁrst 24 h of incubation and remained
Fig. 1. Proteasome enzymatic activities in Raji (A) and Akata (B) cells treated with EBV lytic cycle inducers. Raji cells and EBV-positive Akata cells were
treated with EBV lytic cycle activators as described in Materials and methods. At the indicated times, cell lysates, pre-treated or not with the proteasome
inhibitor MG132, were incubated with the ﬂuorogenic substrates Succ-LLVY-AMC, Z-ARR-AMC, Z-LLE-AMC to evaluate chymotryptic-like, trypsin-
like and caspase-like activities, respectively. The MG132 suppressible activity, considered to be proteasome speciﬁc, was evaluated. Data shown in the bar-
graph are the mean values of triplicate determinations carried out at diﬀerent times after addition of EBV activators to the cells and expressed as
percentage of the activity measured in control cells (time 0) taken as 100%. Standard errors of the mean are shown as error bars.
G. Matusali et al. / Biochemical and Biophysical Research Communications 352 (2007) 947–952 949
similarly low for the entire time frame of the experiment. In
contrast, the activity of TPPII in Akata cells did not show a
signiﬁcant decrement when tested in cells incubated for
24 h with anti IgG. However, measurements carried out
in cells exposed for shorter times to the inducer (Fig 2B),
showed that TPPII activity undergoes approximately a
80% reduction within the ﬁrst six hours of incubation
and thereafter progressively increases to the levels mea-
sured in untreated cells. The discrepancy in the results
reported for the two cell lines might be related to the diﬀer-
ent pattern of EBV lytic gene expression in Raji and in
Akata cells. As VCA induction is detected in Akata cells
as early as 6 h post-treatment [27,29], it is conceivable that
TPPII activity is restored during the late phases of the lytic
In order to clarify whether the reduced activities of the
two proteolytic systems measured in Raji and in Akata
cells were dependent on viral activation and not on the
compounds used in the experiments, EBV-negative Ramos
cells were treated with TGF-b, sodium butyrate and P
(BU)2. Proteasomal and TPPII activities were measured
during the ﬁrst 24 h of incubation when cell viability was
more than 85%. The results obtained showed that protea-
somal activities as well as TPPII activity remained substan-
tially stable over the time frame of the experiment,
indicating that impaired function of the two systems is
dependent on EBV lytic cycle activation (data not shown).
To investigate whether the levels of the proteolytic com-
plexes were also aﬀected by EBV activation, lysates from
Raji and from Akata cells collected at diﬀerent times after
exposure to lytic cycle inducers, were analyzed by Western
blots with antibodies directed to the catalytic or the regula-
tory subunits of the proteasome as well as with antibodies
speciﬁc for the TPPII complex. Fig. 3 illustrates the results
obtained for Raji cells. Polyclonal antibodies recognizing
a/b subunits of the 20S proteasomal core (Fig. 3A) detect
proteins of about 25–30 kDa, whose signal decreased by
diﬀerent extents during the time frame of the experiment.
At 48 h only the larger component was still detectable
and at 72 h all the bands had virtually disappeared. In
Fig. 3B, a Western blot carried out with antibodies detect-
ing S6’ATPase, a subunit of the 19S regulatory complex is
reported. Unlike that observed for the components of the
catalytic core, the level of this subunit increased about
three-fold during the 72 h following EBV induction.
As ubiquitinatio n is required for degradation of most
proteasomal substrates, we tested whether inhibition of
proteasome activity would aﬀect the clearance of ubiquiti-
nated proteins. Fig. 3C shows a representative Western
blot probed with anti-ubiquitin antibodies in which it
appears a time-dependent accumulation of ubiquitin conju-
gates occurring after addition to the cells of lytic cycle acti-
vators. Quantiﬁcation of the signals revealed a 3- to 4-fold
increment of ubiquitinated species after 72 h of incubation
(Fig. 3D). Cell viability at this time, determined by Trypan
blue dye exclusion was higher than 80% and sub-G1 events
in cytoﬂuorimetric cell-cycle analysis did not exceed 25%.
These results therefore, rule out the possibility that the
increment of ubiquitinated proteins was dependent on cell
death or apoptosis (data not shown). As the S6’ subunit
which binds and directs ubiquitinated protei ns to the cata-
lytic chamber, appeared to increase to a similar extent over
the same time, it seems likely that a stimulatory eﬀect is
exerted by ubiquitin-bound substrates on S6’ subunit regu-
lation. The recent ﬁnding that S6’ ATPase activity is great-
ly increased in the presence of ubiquitinated substrates 
supports this ﬁnding.
Fig. 2. Enzymatic activitiy of TPPII in Raji (A) and Akata (B) cells
treated with EBV lytic cycle inducers. Raji (A) and Akata (B) cells were
treated as described in Fig. 1. TPPII activity was measured in the cell
lysates collected at the indicated times, pre-incubated or not with the
TPPII inhibitor AAF-CMK, after addition of the ﬂuorogenic substrate
AAF-AMC. The AAF-CMK suppressible activity, considered to be TPPII
speciﬁc, was evaluated. Data shown in the bar-graph are the mean values
of triplicate determinations carried out at the diﬀerent times and expressed
as percentage of the activity of control cells (time 0) taken as 100%.
Standard errors of the mean are shown as error bars.
Fig. 3. Western blot analysis of Raji cells after exposure to EBV lytic cycle
activating compounds. Raji cells were treated with EBV lytic cycle
activating compounds as described . At diﬀerent times, samples of cell
lysates were processed as described in the Materials and methods. The
blots were probed with (A) anti 20S proteasome a/b subunits (B) anti-S6’
proteasome subunit; (C) anti ubiquitin; (E) anti-TPPII; (F) anti GAPDH.
Speciﬁc signals detected by enhanced chemoluminescence were quantiﬁed
by densitometric analysis. In (D) are reported the densitometric values, in
arbitrary units, of the blot shown in (C). For all blots, one representative
experiment out of three is shown.
950 G. Matusali et al. / Biochemical and Biophysical Research Communications 352 (2007) 947–952
We next evaluated whether decreased enzymatic activity
of the TPPII complex during EBV lytic cycle induction cor-
related with down regulation of TPPII expression. TPPII
antibodies revealed a band of approximately 140 kDa
(Fig. 3E). The intensity of this band transiently increased
during the ﬁrst 24 h of incubation, but consistently dimin-
ished in lysates of cells exposed for longer periods of time
to the inducers. Hybridization of the blots with antibodies
directed to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used to verify that similar amounts of pro-
teins had been loaded onto each gel track (Fig. 3F).
Western blots carried out on Akata cell lysates gave
results similar to those obtained with Raji cells although
diﬀerences in protein expression were less marked, due to
the lower percentage of EBV activation obtained with this
cell line (data not shown).
Taken together, the data here reported indicate that, fol-
lowing EBV lytic cycle induction, the activities of two
major cellular proteolytic systems, the 26S proteasome
and the TPPII complex, are down-regulated in Burkitt’s
lymphoma cells, both at the protein level as well as at the
functional level. We show that impairment of proteasomal
activity is accompanied by a speciﬁc loss of subunits
belonging to the 20S catalytic core. As the EBV lytic cycle
is triggered by diﬀerent inducers in Raji and Akata cells,
the results described are likely to de pend on EBV activa-
tion. Moreover, since expression of EBV late antigens is
prevented in Raji cells, impairment of the cellular proteo-
lytic systems appears to be an early event upon induction
of EBV lytic cycle. We do not know yet, however, by which
mechanisms modulation of speciﬁc subunits of the catalytic
or the regulatory complex occur as a result of EBV lytic
It is well known that cell protein synthesis is blocked by
viral reactivation. It is conceivable that impairment of pro-
tein degradation systems during the early phases of the lytic
cycle might prevent processing of the newly synthesized
viral antigens favoring EBV replication and evasion of
the host immune response.
Forthcoming research aimed at understanding which
viral lytic products interfere with the host proteolytic sys-
tems after EBV activation should have an impact on both
the control of virally infected cells and the development of
new immunization strategies.
This work was partially supported by a contribution of
the ‘‘Istituto Pasteur Fondazione Cenci Bolognetti’’, Uni-
versity of Roma, ‘‘La Sapienza’’ and by grants from the
Italian Ministry of Education, University and Research
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