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ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: http://www.tandfonline.com/loi/kccy20

Viral oncoprotein LMP1 disrupts p53-induced cell

cycle arrest and apoptosis through modulating
K63-linked ubiquitination of p53

Lili Li, Wei Li, Lanbo Xiao, Juan Xu, Xue Chen, Min Tang, Zigang Dong, Qian
Tao & Ya Cao

To cite this article: Lili Li, Wei Li, Lanbo Xiao, Juan Xu, Xue Chen, Min Tang, Zigang Dong, Qian
Tao & Ya Cao (2012) Viral oncoprotein LMP1 disrupts p53-induced cell cycle arrest and apoptosis
through modulating K63-linked ubiquitination of p53, Cell Cycle, 11:12, 2327-2336, DOI: 10.4161/
cc.20771

To link to this article: https://doi.org/10.4161/cc.20771

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Published online: 15 Jun 2012.

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 RepOrT rePort
Cell Cycle 11:12, 2327-2336; June 15, 2012; © 2012 Landes Bioscience

Viral oncoprotein LMP1 disrupts p53-induced cell

cycle arrest and apoptosis through modulating
K63-linked ubiquitination of p53
Lili Li,1-3,† Wei Li,1,2,† Lanbo Xiao,1,2 Juan Xu,1,2 Xue Chen,1,2 Min Tang,1,2 Zigang Dong,4 Qian Tao3 and Ya Cao1,2,*
1
Cancer Research Institute; XiangYa School of Medicine; Central South University; Changsha, China; 2Carcinogenesis and Invasion Key Laboratory of Education Ministry
of China; Central South University; Changsha, China; 3Cancer Epigenetics Laboratory; Department of Clinical Oncology; State Key Laboratory of Oncology in South China;
Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences; The Chinese University of Hong Kong and CUHK Shenzhen Research Institute; Hong Kong, China;
4
The Hormel Institute; University of Minnesota; Austin, MN USA

†
These authors contributed equally to this work.

Keywords: p53, ubiquitination, K63, Epstein-Barr virus, LMP1

Disruption of the gatekeeper p53 tumor suppressor is involved in various virus-associated tumorigeneses, with aberrant
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ubiquitination as the major cause of p53 abnormalities in virus-associated tumors. Of note, wild-type p53 is accumulated
in Epstein-Barr virus (EBV)-associated tumors, especially in nasopharyngeal carcinoma (NPC). We have previously
identified that p53 is accumulated and phosphorylated by EBV oncoprotein latent membrane protein 1 (LMP1) in NPC.
Here, we further found that LMP1 promoted p53 accumulation via two distinct ubiquitin modifications. LMP1 promoted

© 2012 Landes Bioscience.

p53 stability and accumulation by suppressing K48-linked ubiquitination of p53 mediated by E3 ligase MDM2, which is
associated with its phosphorylation at Ser20, while increasing the levels of total cellular ubiquitinated p53. LMP1 also
induced K63-linked ubiquitination of p53 by interacting with tumor necrosis factor receptor-associated factor 2 (TRAF2),
thus contributing to p53 accumulation. Furthermore, LMP1 rescued tumor cell apoptosis and cell cycle arrest mediated

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by K63-linked ubiquitination of p53. Collectively, these results demonstrate aberrant ubiquitin modifications of p53
and its biological functions by viral protein LMP1, which has broad implications to the pathogenesis of multiple EBV-
associated tumors.

Introduction Epstein-Barr virus (EBV), a human herpesvirus, is classically

p53, acting as a critical gatekeeper tumor suppressor, is involved
in multiple virus-mediated tumorigeneses.1-3 Metabolically
stable and intact p53 exerts its physiological functions in virus-
induced tumorigenesis,4-8 including transcription regulation
and DNA damage induction,9-12 requiring posttranslational
modifications, such as phosphorylation, ubiquitination and
acetylation. Disruption of the ubiquitin proteasome pathway
is the major cause of p53 abnormities in virus-induced tumori-
genesis. Some viral proteins, such as simian virus 40 (SV40) T
antigen, E1B 55-kDa and human papillomavirus (HPV) E6
protein, deregulate p53 activity and stability via an ubiquitina-
tion system.2,13-15 Lys48 (UbK48) is often used for targeting
substrate protein to 26S proteasome-dependent degradation,
whereas Lys63 (UbK63) plays a key role in nonproteolytic
events, including DNA repair, endocytosis and kinase-medi-
ated signaling responses.16,17 E3 ligase MDM2 targeting p53
for K48-linked proteasomal degradation is well defined,18-20 but
less is known regarding the K63-linked ubiquitination of p53.
implicated in multiple tumorigeneses, including Burkitt lym-
phoma, B-cell lymphoproliferative diseases, nasal NK/T-cell lym-
phoma and nasopharyngeal carcinoma (NPC).21,22 Although two
alternative infection states exist, latency and lytic, EBV-associated
tumors are predominantly of latent infection. Importantly, wild-
type p53 plays a crucial role in maintaining EBV latency by express-
ing excessive p53 over EBV immediate-early (IE) protein BZLF1
(Z), thus suppressing the switch from latent to lytic phase.23-25
Ubiquitin-mediated degradation of p53 by BZLF1 in the middle
and late stages of EBV lytic infection has been identified.25,26
As one of the EBV encoded-proteins with oncogenic proper-
ties, EBV latent membrane protein 1 (LMP1) activates multiple
signaling pathways, such as MAPK, NFκB, JAK/STAT, p53 etc.,
through recruiting tumor necrosis factor receptor type 1-asso-
ciated DEATH domain protein (TRADD) and TNF receptor-
associated factors (TRAFs) at the C-terminal activation region
(CTAR) 1 or CTAR 2, thus contributing to EBV-mediated
tumorigenesis.27,28 Unlike other tumors, wild-type Tp53 is accu-
mulated in EBV-associated tumors especially in NPC,8,29‑31
displaying a correlation with LMP1 in clinical samples.32-34

*Correspondence to: Ya Cao; Email: ycao98@vip.sina.com

Submitted: 02/08/12; Revised: 05/14/12; Accepted: 05/15/12
http://dx.doi.org/10.4161/cc.20771

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© 2012 Landes Bioscience.

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Figure 1. LMP1 upregulated p53 expression and its activity. (A) LMP1 upregulated endogenous p53 and MDM2 expression in a dose-dependent man-
ner. Increasing amounts of Flag-LMP1 were transfected into MCF7 cells. (B) LMP1 increased p53, MDM2, p21 expression and p53 Ser20 phosphoryla-
tion. Flag-LMP1 and GFP-p53 expression plasmids were transfected into H1299 and Saos2 cells as indicated. (C) LMP1 promoted p53-mediated MDM2
upregulation and its phosphorylation. GFP-p53, HA-MDM2 and different amount of Flag-LMP1 were transfected into H1299 cells. (D) LMP1 aborted
MDM2-mediated the downregulation of p53 and p53 Ser20. GFP-p53, Flag-LMP1 and different amount of HA-MDM2 were transfected into H1299 cells
as indicated. After transfection, cell lysates were prepared and analyzed by western blot with the indicated antibodies.

However, the mechanism and function of aberrant p53 accumu-
lation/stability in EBV latency have been controversial, though
few mutations were reported. Our previous studies have identi-
fied that LMP1 upregulated p53 expression and its phosphoryla-
tion at Ser15, Ser20, Ser392 and Thr81 through MAPK signaling
pathway, which is responsible for its stability and activity.12,35,36
However, the mechanism of p53 accumulation by LMP1 via
ubiquitin-proteasome pathway needs further investigation.
Here, we studied p53 accumulation/stability via different
ubiquitin modifications by LMP1 and its related biological func-
tions. We found that LMP1 inhibited K48-linked ubiquitina-
tion of p53 by decreasing the interaction of p53 and MDM2.
Meanwhile, LMP1 promoted K63-linked ubiquitination of p53
by increasing the interaction of p53 and TRAF2. Furthermore,
LMP1 rescued cell cycle arrest and apoptosis of tumor cells
induced by K63-linked ubiquitination of p53, contributing to
EBV-associated tumorigenesis.
Results
Viral protein LMP1 increases p53 accumulation and activity.
As the presence of mutant p53 in most of NPC cell lines unlike
its physiological status,29,31 we firstly evaluated the effect of EBV
LMP1 on p53 expression and activity by ectopic expressing LMP1
and/or p53 in p53-intact and p53-null cells. Results revealed that
LMP1 could upregulate the expression of endogenous p53 and
MDM2 in a dose-dependent manner in MCF7 cells (Fig. 1A). In
LMP1-expressing H1299 and Saos2 cells, p53 Ser20 phosphor-
ylation, important for p53 stability and activity, as well as the
expression of its downstream target genes, MDM2 and p21, were
increased upon enhanced expression of p53 (Fig. 1B). These data
suggest that LMP1 promotes the expression of p53 and its target
genes, completely consistent with our previous findings in NPC
cell lines with partly wild-type p53.
As MDM2 is recognized to mediate p53 degradation, we
next assessed p53 expression via ectopically transfected with
LMP1, p53 and MDM2 in H1299 cells. Our data showed that
LMP1 could dose-dependently upregulate p53 and MDM2 pro-
tein level as well as p53 Ser20 phosphorylation (Fig. 1C). Upon
increased MDM2 expression, p53 downregulation was observed,
as expected, together with the decrease of p53 Ser20 phosphory-
lation, while LMP1 restored p53 expression and its phosphoryla-
tion (Fig. 1D). These results suggest that LMP1 increases p53
accumulation and activity.

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Figure 2. LMP1 inhibited p53 and MDM2 degradation but increased p53 ubiquitination. (A and B) Effects of CHX on p53 and MDM2 stability mediated
by LMP1. GFP-p53 or HA-MDM2 and Flag-LMP1 were transfected into H1299 cells and then treated with CHX (20 μg/ml) for the indicated times. p53
and MDM2 expression levels were determined by western blot with the indicated antibodies. The levels of p53 and MDM2 protein were quantified
by densitometry. (C) The relative binding affinity of p53 and MDM2 or LMP1 by immunoprecipitation in H1299 cell line. Cells were transfected with
GFP-p53, HA-MDM2 and Flag-LMP1. Cell lysates were immunoprecipitated with GFP, HA or Flag antibodies and then analyzed their binding affinity
by western blot. (D) LMP1 promoted p53 ubiquitination. Different expression vectors, including p53, Ub and LMP1 as indicated, were transfected into
H1299 cells and then treated with MG132 for 6 h. p53 ubiquitination was monitored in immunoblots performed on Ni-NTA purified proteins. To show
the expression levels of p53 protein, the same cell lysates were subjected to western blot.

LMP1 promotes p53 stability and ubiquitination. To
demonstrate p53 stability mediated by LMP1, the effects of
LMP1 on the half-life of p53 and MDM2 using cycloheximide
(CHX) chase were examined. We found that LMP1 signifi-
cantly extended the half-life of p53 from about 35 min to over
90 min as well as that of MDM2 from about 40 min to over
90 min (Fig. 2A and B), suggesting that LMP1 increased the
stability of p53 and MDM2 simultaneously. We next exam-
ined the possible interaction of LMP1 with p53 and MDM2.
Results revealed that LMP1 reduced the interaction of p53 to
MDM2 (Fig. 2C, upper), while no binding with p53 was found
(Fig. 2C, lower).
We further evaluated whether ubiquitin-dependent protea-
some system was involved in LMP-mediated p53 stability. In
NP69-LMP1 stable NPC cells, we found that LMP1 obviously
increased p53 ubiquitination in the presence and absence of a
proteasome inhibitor, MG132 (Fig. S1). Ubiquitination assay
via Ni-NTA (nickel-nitrilotriacetic acid) purification further

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© 2012 Landes Bioscience.

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Figure 3. The suppression of MDM2-mediated p53 ubiquitination by LMP1 is associated with p53 Ser20 phosphorylation. (A) LMP1 inhibited MDM2-
mediated p53 ubiquitination. The expression plasmids, including GFP-p53, HA-MDM2, His-Ub and Flag-LMP1 were transfected into H1299 cells as
indicated and then treated with MG132 for 6 h. p53 ubiquitination was performed by Ni-NTA pull-down analysis. The same cell lysates were subjected
to western blot to show the expression levels of these proteins. (B) LMP1 impeded destabilized p53 by p53 Ala20. H1299 cells were transfected with
expression plasmids for GFP-p53, GFP-p53 Ala20, GFP-p53 Asp 20 and Flag-LMP1 as indicated. (C) LMP1 rescued p53 stability disrupted by p53 Ala20 in
a dose-dependent manner. GFP-p53, GFP-p53 Ala20, GFP-p53 Asp 20, HA-MDM2 and different amount of Flag-LMP1 were transfected into H1299 cells.
After transfection, cell lysates were prepared and analyzed by western blot with the indicated antibodies. (D) LMP1 destabilized p53 Ala20 through the
inhibition of its ubiquitination. p53, its mutants, Ub and LMP1 were transfected into H1299 cells as indicated and then treated with MG132 for 6 h. p53
ubiquitination was performed by immunopercipitation with anti-GFP antibody. To show the expression levels of p53, MDM2 and LMP1 protein, the
same cell lysates were subjected to western blot.

showed the increase of p53 ubiquitination by LMP1 in H1299
cells co-transfected with His-Ub, p53 and LMP1, which
is significantly enhanced by MG132 treatment (Fig. 2D).
These results indicate that LMP1 promotes p53 stability and
ubiquitination.
LMP1 inhibits MDM2-mediated p53 ubiquitination associ-
ated with p53 Ser20 phosphorylation. As MDM2 is a p53-spe-
cific E3 ubiquitin ligase, we next investigated whether MDM2
was required for increased p53 ubiquitination by LMP1. Results
showed that p53 ubiquitination was increased with ectopic

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Figure 4. LMP1 induced K63-linked ubiquitination of p53 via its C-terminal domain. (A) LMP1 dramatically increased K63-linked ubiquitination of p53.
H1299 cells were transfected with the His-Ub (wt, K48, K63), GFP-p53 and Flag-LMP1 plasmids. p53 was immunoprecipitated with anti-GFP antibody
and the precipitated materials were subjected to immunoblot. (B) LMP1 increased K63-linked ubiquitination of p53 in the presence of E3 ligase MDM2.
Cells were transfected with the indicated plasmids. p53 ubiquitination was performed by immunoprecipitated anti-GFP antibody, the same cell lysates
were subjected to western blot to show the expression levels of p53, MDM2 and LMP1 protein. (C) LMP1 promoted p53 accumulation via its C-terminal
domain. H1299 cells were transfected with different expression vectors, including Flag-LMP1, its mutants and GFP-p53. After transfection, cell lysates
were prepared and analyzed by western blot with the indicated antibodies. (D) LMP1 induced K63 ubiquitination of p53 via its C-terminal domain.
Different expression vectors, including GFP-p53, His-UbK63, Flag-LMP1 and its mutant were transfected into H1299 cells. p53 ubiquitination was per-
formed on Ni-NTA pull-down ubiquitination analysis.

expression of Ub, p53 and MDM2 in H1299 cells, consistent
with the previous studies, but was reduced in presence of LMP1
(Fig. 3A), suggesting that E3 ligase MDM2 is not responsible for
increased p53 ubiquitination by LMP1.
Since phosphorylation of p53 at Ser20 increases p53 stability
by disrupting the interaction between p53 and MDM2, whether
p53 Ser20 is involved in LMP1-mediated p53 stability and ubiq-
uitination was further addressed. We found that substitution of
Ser20 by Ala (p53Ala20) significantly reduced p53 expression,
as well as its target genes p21 and MDM2, through using p53
Asp20 as a control mimicking constitutive phosphorylation of
p53 Ser20, but p53 was restored by LMP1 (Fig. 3B). Moreover,
LMP1 increased p53 expression in a dose-dependent man-
ner through ectopic expression of both MDM2 and p53 Ala20
(Fig. 3C), while no p53 ubiquitination was observed even after
MG132 treatment (Fig. 3D), indicating that the inhibition of
MDM2-mediated p53 ubiquitination by LMP1 is associated
with p53 Ser20 phosphorylation.
LMP1 increases K63-linked ubiquitination of p53 through
its C-terminal domain. Given the increase of p53 ubiquitination
and accumulation in LMP1-expressing cells, distinct lysines of
Ub linked to p53 were further analyzed. Ubiquitination assay
showed that LMP1 suppressed K48-linked ubiquitination of p53,
in line with the above findings, but increased K63-linked ubiq-
uitination of p53 and further resulted in the total increase of p53
ubiquitination (Fig. 4A). In addition, LMP1 significantly inhib-
ited K48-linked ubiquitination of p53 mediated by MDM2;
meanwhile, K63-linked ubiquitin chains were still increased
(Fig. 4B), suggesting that increased p53 ubiquitination by LMP1
is mainly K63-linked ubiquitin chains.

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Figure 5. TRAF2 is required for K63-linked ubiquitination of p53 by LMP1. (A) K63-linked ubiquitination of p53 by LMP1 is associated with TRAF2 but
not TRAF6. H1299 cells were transfected with different expression vectors, including siRNA-TRAF6, siRNA-TRAF2, His-UbK63, GFP-p53 and Flag-LMP1
as indicated. After transfection, cell lysates were prepared and analyzed for expression of p53. (B) LMP1 promoted the interaction of TRAF2 with p53.

© 2012 Landes Bioscience.

H1299 cells were transfected with GFP-p53, TARF2, His-UbK63 and Flag-LMP1. Cell lysates were immunoprecipitated with TRAF2, and then binding af-
finity with p53 was analyzed by western blot. (C) LMP1 induced K63-linked ubiquitination of p53. Different expression vectors, including GFP-p53, His-
UbK63, TRAF2 and Flag-LMP1, were transfected into H1299 cells. p53 ubiquitination was performed by immunopercipitation with anti-GFP antibody.

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We next investigated the functional domain of LMP1 for
K63-linked ubiquitination of p53. Western blot analysis showed
that wild-type LMP1 as well as its CTAR1 and CTAR2 mutants
promoted p53 accumulation but not for its C-terminal deletion
mutant (Fig. 4C). Ubiquitination assay further showed that
LMP1 significantly enhanced K63-linked ubiquitination of p53
via its C-terminal domain (Fig. 4D), consistent with its expres-
sion. Thus, the C terminus of LMP1 is responsible for K63-
linked ubiquitination of p53 and its stability.
TRAF2 is involved in LMP1-mediated K63-linked ubiqui-
tination of p53. Both TRAF2 and TRAF6 have been reported
to act for K63-linked ubiquitination and also to be activated by
LMP1; we next evaluated the possible E3 ligase for K63-linked
ubiquitination of p53 by LMP1. By RNAi-mediated silencing of
TRAF2 or TRAF6, results showed that knockdown of TRAF2
significantly suppressed p53 expression in the presence and
absence of LMP1, but no such effect was observed in TRAF6-
siRNA-transfected cells (Fig. 5A).
We next examined the interaction between the TRAF2 and
p53 mediated by LMP1. Co-immunoprecipitation assay showed
that LMP1 enhanced the binding of TRAF2 and p53 linked
with K63 ubiquitin chains (Fig. 5B). Furthermore, the assembly
of K63-linked ubiquitination was detected only with the coexis-
tence of p53, TRAF2 and LMP1 (Fig. 5C). These results indicate
that LMP1 induces K63-linked ubiquitination of p53 by TRAF2.
LMP1 rescues tumor cell apoptosis and cell cycle arrest by
K63-linked ubiquitination of p53. We further investigated the
biological effects of K63-linked ubiquitination of p53 on apopto-
sis and cell cycle by LMP1. Assessment for activation of caspases
showed that the cleavage of caspase-3, -8 and -9 proenzymes to
their active forms were significantly reduced in the presence of
LMP1 together with a decrease of cleaved PARP (Fig. 6A). A sig-
nificant increase (> 14-fold) of the sub-G1 population induced by
K63-linked ubiquitination of p53 was observed, while only 5%
of cells displayed signs of apoptotic death in LMP1-expressing
tumor cells as measured by flow cytometry (Fig. 6C), indicating
LMP1 inhibits tumor cell apoptosis by K63-linked ubiquitina-
tion of p53.
The distributions of cell cycle phase by K63-linked ubiquiti-
nation of p53 in the presence and absence of LMP1 were exam-
ined. Flow cytometry analysis demonstrated that p53-induced
G1/S and G2 /M cell cycle arrest were abrogated in tumor cells
that ectopically expressed LMP1 (Fig. 6B). Consistently, cyclin
inhibitors p21 and p27 levels were decreased, while key cyclins
and cyclin-dependent kinases (CDKs) in cell cycle, such as cyclin
D1-CDK4, cyclin A-CDK2 and cyclin B1-CDK1, were increased
by LMP1 (Fig. 6D). These results indicate that LMP1 disrupts
tumor cell apoptosis and cell cycle arrest mediated by K63-linked
ubiquitination of p53.
Discussion
The present study uncovers protein ubiquitination as one of the
mechanisms in regulating p53 accumulation/stability by LMP1,
thus contributing to EBV latency in EBV-associated tumorigen-
esis. As one of the EBV encoded proteins with tumorigenicity,
LMP1 promotes wild-type p53 accumulation/stability and tran-
scriptional activity through distinctly regulating p53 ubiquitin

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Figure 6. LMP1 suppressed tumor cell apoptosis and cell cycle arrest induced by K63-linked ubiquitination of p53. (A and B) Expression of regulatory
proteins of apoptosis and cell cycle by western blot. H1299 cells were transfected with GFP-p53, TARF2, His-UbK63 and Flag-LMP1 as indicated and
then monitored for protein expression involved in induction of apoptosis and cell cycle arrest. (C and D) Flow cytometric analysis of cell cycle arrest
and apoptosis. p53, UbK63, TRAF2 and LMP1, were transfected into H1299 cells. Apoptotic rate and the percentages of cell cycle phases were deter-
mined as described in (C and D).

K48 or K63 linkage, resulting in uncontrolled cellular prolifera-
tion without inducing apoptosis. Thus, this study for the first
time elucidates the aberrant ubiquitin modifications of p53 and
its biological functions by viral protein LMP1 in EBV latency.
The previous concept considered that p53 inactivation,
through mutation, degradation or binding with viral proteins,
is involved in virus-induced tumorigenesis.37,38 One of the clas-
sical mechanisms of p53 inactivation is binding with SV40
large T antigen, with the increase of p53 stability.39 Adenovirus
employs a similar strategy as SV40 to inactivate p53.14 In HPV
E6-transformed cells, p53 is destabilized and virtually elimi-
nated through enhancing its ubiquitination.13,15 These indicate
that disrupted p53 plays a critical role in virus-induced trans-
formation. Remarkably, increasing evidences showed that p53
is constitutively active in virus-induced tumorigenesis.7-10 Our
previous studies demonstrated that LMP1 increased p53 accu-
mulation and transcriptional activity via promoting its phos-
phorylation in NPC cells.35 We also found that activated p53 by
LMP1 as a transcript factor upregulated survivin expression and
activation, thus resulting in cell cycle progression and apoptosis
inhibition in NPC pathogenesis.40 Here, using p53-null cells as
models, we found that LMP1 increased p53 accumulation and
activity as well as its stability, in line with our previous and oth-
ers studies.
The disruption of p53 ubiquitination during EBV latent
and lytic phase by other EBV-encoded proteins besides LMP1
has been reported. For example, during EBV latent infection,
Epstein-Barr nuclear antigen 1 (EBNA1) protein inhibits USP7
deubiquitinase and thus drives p53 ubiquitination.41 During EBV
lytic infection, p53 is ubiquitinated by BZLF1-associated E3
ligase independently of MDM2.26 BZLF1 protein regulates p53
concordantly through transient induction of p53 for the initia-
tion of viral productive replication at the early stages, and degra-
dation of p53 for the establishment of a S phase-like environment
at the later stages preferable for viral replication,25 indicating the
complexity of p53 modulation in EBV-induced tumorigenesis.

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 In summary, our study identifies that
EBV latent oncoprotein LMP1 promotes
p53 accumulation/stability through pro-
moting K63-linked ubiquitination, thus
rescuing tumor cell apoptosis and cell
cycle arrest (Fig. 7). This work extends
our understanding of aberrant ubiquitin
modifications of p53 by LMP1 facilitating
EBV latency.

Materials and Methods

Cell lines and transfection. Human lung

adenocarcinoma cell line H1299, osteo-
sarcoma cell line Saos2, breast adenocar-
Figure 7. Proposed model for p53 accumulation/stability by LMP1 in EBV latency.
cinoma cell line MCF7 cells were grown in
RPMI medium supplemented with 10%
MDM2 has been identified to tightly control p53 stability by fetal bovine serum in a 37°C incubator with 5% CO2. NP69,
both self- and SCF(β-TRCP)-dependent ubiquitination.42 In NP69-pLNSX and NP69-LMP1 nasopharyngeal epithelial cell
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this study, we illustrated distinct regulatory mechanisms of p53 lines were grown in keratinocyte-SFM medium (Invitrogen).56,57
ubiquitination mediated by EBV latent oncoprotein LMP1. We These cell lines were obtained either from the American Type
found that LMP1 suppressed K48-linked ubiquitination of p53 Culture Collection or from our collaborators with no authen-
mediated by E3 ligase MDM2, associated with phosphorylation tication performed. All transfections were performed with
© 2012 Landes Bioscience.
at Ser20, while it increased K63-linked ubiquitination of p53. LipofectamineTM 2000 transfection reagent (Invitrogen) accord-
Recently, ubiquitination linkage through K63-linked ubiq- ing to the manufacturer’s protocol.
uitin chains serving proteasome-independent functions has Plasmids and reagents. Flag-LMP1 wild type (WT), 1–231-

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attracted more attention, including protein-protein interactions, only (CTAR1), 187–351-only (CTAR2) plasmids and TRAF2
activation of signaling molecules and DNA damage responses. expression plasmids were kindly provided by Dr. Kenneth M.
For instance, A20 zinc finger 4 (ZnF4) activated NFκB signaling Izumi (Brigham and Women’s Hospital). GFP-p53, His-Ub and
through binding with K63-linked poly-Ub.43,44 CYLD inhibited HA-MDM2 were gifts of Dr. Qian Wu (Xiamen University)
Wnt/β-Catenin signaling through K63-linked ubiquitination of and have been described previously in reference 12 and 58. His-
Dishevelled (Dvl).45 Phosphorylation-dependent Daxx with K63- UbK48-only and His-UbK63-only were generated in our lab and
linked ubiquitination functions as a molecular switch to initiate verified by sequencing. Cycloheximide (CHX) and MG132 were
and amplify the stress kinase response in the TNFα signaling purchased from Sigma.
pathway.46 Some members of TRAFs have been identified to act Antibodies. Anti-CyclinD1 (sc-450) and anti-CDK4
for K63-linked ubiquitination.47,48 IKK is activated by TRAF6, (sc-260), anti-CyclinA (sc-596), anti-CDK2 (sc-163), anti-
involving K63-linked polyUb chains, but not for proteasomal CyclinB1 (sc-7393), anti-CDK1 (sc-954), anti-p27 (sc-1641),
degradation.49 There is evidence that TRAF6 acting as E3 ligase anti-TRAF2 (sc-876), anti-TRAF6 (sc-8409), anti-His (sc-
is required for LMP1-stimulated K63-linked ubiquitination of 804), anti-HA (sc-7392), anti-GFP (sc-9996) anti-rabbit
IRF7 and enhanced transcriptional activity.50,51 Our results dem- IgG-HRP (sc-2004), anti-mouse IgG-HRP (sc-2005), anti-
onstrated that TRAF2 but not TRAF6 was responsible for K63- goat IgG-HRP (sc-2020) and normal mouse/rabbit IgG were
linked ubiquitination of p53 by LMP1, thus resulting in p53 purchased from Santa Cruz Biotechnology. Anti-p21 (2947),
accumulation/stability in EBV latency, which also provides clues anti-Caspase 3 (9665), anti-Caspase 8 (9746), anti-Caspase 9
for proteasome inhibitor-mediated anticancer therapy.52 (9502) and anti-Cleaved PARP (9541), were obtained from Cell
Disruption of p53 function in virus-induced tumorigenesis Signaling Technology. Mouse anti-Flag M2 and β-actin (Sigma
establishes cellular conditions appropriate for persistent latent Aldrich) antibodies were also used. TRAF2 siRNA (sc-29509)
infection and periodic viral replication.23,53,54 Our findings of and TRAF6 siRNA (sc-36717) were purchased from Santa Cruz
the abrogation of p53-induced apoptosis and cell cycle arrest by Biotechnology.
LMP1 supported this role. Through disrupting host ubiquitin- Immunoprecipitation and western blot. Cells were har-
proteasome system, aberrant p53 activation by LMP1 facilitates vested in NP40 lysis buffer (50 mmol/L TRIS-HCl, pH 8.0;
EBV latency-associated tumorigenesis. The dilemma between 150 mmol/L NaCl; 0.5% NP40) with protease inhibitor cocktail
virus infection and host cell responses has always being strug- (Roche) and phosphatase inhibitors (Sigma Aldrich). For immu-
gled, accompanied by host cell DNA damage responses.55 Further noprecipitation, cell lysates were incubated with specific antibod-
studies are needed to explore the role of K63-linked ubiquitina- ies, followed by incubation of protein A/G agarose (Amersham
tion of p53 in DNA damage responses for EBV latency-associated Biosciences AB). The precipitated proteins were eluted from
malignancies. resuspending the beads before western blot. For western blot, cell

2334 Cell Cycle Volume 11 Issue 12

 lysates were subjected to SDS-PAGE and blotted with specific
antibodies as indicated above. Immunoreactive bands were visu-
alized using ECL chemiluminescence reagents (Pierce Chemical
Co.,) according to the manufacturer’s protocol.
Protein stability analysis. For p53, MDM2 and LMP1 pro-
tein stability experiments, 20 μg/ml CHX was added to cells to
inhibit protein synthesis. Subsequent time points were incubated
in medium containing CHX for 0, 20, 40, 60 or 90 min as indi-
cated. Cells were then processed as described above for western
blot and quantitated by densitometric measurement.
Ubiquitination assays. H1299 cells were co-transfected with
His-Ub, His-UbK48, His-UbK63 and other expression plasmids
as indicated. Forty hours after the transfection, the cells were
treated with 10 μM of MG132 (Sigma Aldrich) for 6 h prior to
harvest. Cells were split into two aliquots, one for immunoblot
and the other for ubiquitination assays. Cell lysates were affin-
ity purified with Ni-NTA-agarose beads (Qiagen) or incubated
with specific antibodies targeting p53 or MDM2 and analyzed
by immunoblot, as described previously.
Downloaded by [36.73.85.115] at 22:42 02 January 2018
His-UbK63 and Flag-LMP1 expression plasmids. After trans-
fection, cells were fixed with 70% ethanol and incubated in
DNase-free RNase A (50 mg/ml) at 37°C for 30 min, then
immunostained with propidium iodide (Sigma, 50 mg/ml) for
30 min at 4°C against light. DNA was stained with propidium
iodide and the sub-G1 fraction was measured by flow cytometry.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. George Tsao for some cell lines. This proj-
ect was supported by National Basic Research Program (No.
2011CB504300), Key Program of National Natural Science
Foundation of China (No. 3090101), Joint Research Fund for
Hong Kong and Macao Young Scholars (No. 81028012) and
National Natural Science Foundation (No. 30801337).
Supplemental Materials

Flow cytometry for apoptosis and cell cycle analyses. Supplemental materials may be found here:
Cells were co-transfected with GFP-p53, pcDNA3.1-TRAF2, www.landesbioscience.com/journals/cc/article/20771
11. Hess RD, Brandner G. DNA damage-inducible 21. Tao Q, Young LS, Woodman CB, Murray PG.
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