The Hsp70 inhibitor 2-phenylethynesulfonamide inhibits replication and carcinogenicity of Epstein–Barr virus by inhibiting the molecular chaperone function of Hsp70
Wang et al. Cell Death and Disease
The Hsp70 inhibitor 2-phenylethynesulfonamide inhibits replication and carcinogenicity of Epstein-Barr virus by inhibiting the molecular chaperone function of Hsp70
Huan Wang 0
Lang Bu 0 1
Chao Wang 0
Yaqian Zhang 0
Heng Zhou 0
Xi Zhang 2
Wei Guo 3
Cong Long 0
Deyin Guo 1
Xiaoping Sun 4
0 Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University , Wuhan 430071 , China
1 School of Medicine (Shenzhen), Sun Yat-sen University , Guangzhou 510080 , China
2 Second Clinical College of Wuhan University , Wuhan 430071 , China
3 Department of Pathology and Physiology, School of Basic Medical Sciences, Wuhan University , Wuhan 430071 , China
4 The State Key Laboratory of Virology, Hubei Province Key Laboratory of Allergy and Immune-related Diseases, Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University , Wuhan 430071 , China
Epstein-Barr virus (EBV) can infect cells in latent and lytic period and cause serious disease. Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for the maintenance of the EBV DNA episome, replication and transcription. 2phenylethynesulfonamide (PES) is a small molecular inhibitor of Heat shock protein 70 (Hsp70), which can interact with Hsp70 and disrupts its association with co-chaperones and substrate proteins of Hsp70. In our study, we found that PES could decrease the expression of EBNA1, which is independent of effects on EBNA1 transcription or proteasomal degradation pathway. The central glycine-alanine repeats domain was not required for inhibition of EBNA1 expression by PES. Also, PES could reduce the amount of intracellular EBV genomic DNA. PES inhibited proliferation and migration but induced cell cycle arrest and apoptosis of EBV positive cells. In addition, silencing of Hsp70 decreased expression of EBNA1 and the amounts of intracellular EBV genomic DNA, and PES increased this effect on a dose-dependent manner. On the contrast, over-expression of Hsp70 enhanced the expression of EBNA1 and the amounts of intracellular EBV genomic DNA, but PES inhibited this effect on a dose-dependent manner. Furthermore, Hsp70 interacted with EBNA1 but PES interfered this interaction. Our results indicate that PES suppresses replication and carcinogenicity of Epstein-Barr virus via inhibiting the molecular chaperone function of Hsp70.
Epstein?Barr virus (EBV), a human ?-herpesvirus, is an
obligate human pathogen that can infect cells in viral
latent period and lytic period. In the vast majority of
adult population worldwide, EBV can cause a persistent
latent infection for life, but at most cases it is well
controlled1,2. Nevertheless, EBV infection can result in
serious disease such as infectious mononucleosis (IM),
nasopharyngeal carcinoma (NPC), certain gastric
carcinomas, lymphoproliferative disease, Burkitt and Hodgkin
lymphoma3?7. EBV can cause three types of latent
infection, termed as type I, II, and III latency. While Type I or
Type III latency are observed in Burkitt?s lymphoma cells,
type II latency is found in nasopharyngeal or Hodgkin?s
Epstein?Barr virus nuclear antigen 1 (EBNA1) is the
only protein of EBV-encoded proteins in all forms of
EBV-infected cells2,11. EBNA1 is essential for the
maintenance of the EBV DNA episome, replication,
transcription, and postmitotic EBV genome segregation,
which are important processes for viral persistence and
related oncogenic potential12,13. The C-terminal domain
of EBNA1 binds to the viral origin of plasmid replication
(OriP), which is required for episome maintenance
and DNA replication13,14. The N-terminal domain of
EBNA1 tethers EBV episomes to host cellular mitotic
chromosomes and interphase chromatins, which is
necessary for the persistence of the episome in
propagating cells14?16. The central glycine?alanine repeat (GAr)
domain of EBNA1 can suppress the translation of its own
mRNA and play a key role in the mechanism of immune
Heat shock proteins (Hsps) are highly-preserved
proteins. The Hsps efficiently stable unfolded or misfolded
peptides, repair denatured proteins and prevent the
accumulation of improperly folded or denatured proteins,
thereby protecting normal cells against various harmful
stimuli19?21. Hsp70 is an ATP-dependent chaperone
involving in co-translational and posttranslational protein
folding22. Hsp70 maintains intracellular homeostasis via
binding improperly folded polypeptides, refolds these
clients by performing the cycles of co-chaperone
accelerated ATP hydrolysis, and then transfers them to Hsp90.
In this way, Hsp70 promotes protein transports or
posttranslational modifications, and targets improperly folded
substrates for degradation 23,24.
2-phenylethynesulfonamide (PES), originally identified
as a molecule that inhibits p53 binding to mitochondrion,
was then shown to be a selective Hsp70 function
inhibitor25. Specifically, PES acts on the carboxyterminal
substrate-binding domain of Hsp70 and disrupts its
association with co-chaperones and substrate proteins of
Hsp7026,27. PES-mediated cell death of tumor cells is
associated with the accumulation of misfolded proteins,
the impairment of autophagic processes, and the
inhibition of lysosomal functions 26,28,29.
In this present study, our results indicate that PES
inhibits proliferation and migration and causes cell cycle
arrest of EBV-positive cells. PES induces apoptosis by
inhibiting autophagy in HONE1/Akata and HK1/Akata
cells. However, PES increases the expression of LMP1,
which may induce apoptosis in B95-8 cells by activating
caspase through NF-?B pathway. In addition, PES
decreases EBNA1 expression and reduces lytic replication
of EBV. Meanwhile, our findings illustrate that PES
decreases viral protein and viral genomic DNA via
inhibiting the function of Hsp70. Furthermore, our results
show that Hsp70 interacts with EBNA1 but PES inhibits
this interaction. Finally, PES significantly inhibits the
growth of xenografted tumor induced by HONE1/Akata
cells in BALB/c nude mice.
PES decreases EBNA1 expression independent of effects on EBNA1 transcription or proteasomal degradation
To determine whether PES alters the expression level of
EBNA1, a variety of types of latently infected EBV-positive
cells were treated with vehicle drug control or PES. PES
decreased EBNA1 expression in every EBV-infected cell
line examined (Fig. 1a). As expected, expressions of XIAP
and c-IAP1 (obligate Hsp70 clients, which can serve as
biomarkers to monitor Hsp70 inhibition30,31) were also
decreased, whereas Hsp70 expression was not reduced.
This confirmed that PES can inhibit the function of
Hsp70. In addition, the expression level of LMP1, which is
another EBV latent protein, was not decreased, suggesting
that the inhibitory effect of PES on EBNA1 was specific
(Fig. 1a). As shown in Fig. 1b, the Hsp70 natural
expression levels in HONE1/Akata and HK1/Akata cells were
higher than the level in B95-8 cells, which may lead to the
high sensitivity to EBNA1 inhibition by lower dose of PES
in B95-8 cells. To further determine if PES decreases
EBNA1 expression, HONE1/Akata cells were transfected
with pSG5-LMP1. HONE1/Akata, CNE1 and HeLa cells
were transfected with pSG5-EBNA1. These cells were
then treated with PES. As shown in Fig. 1c and e, PES
significantly decreased the expression of transfected
pSG5-EBNA1, whereas expression of pSG5-LMP1 was
increased. PES reduced the expression level of EBNA1,
however, the mRNA levels of EBNA1 were not
significantly decreased after PES treatment, suggesting that
PES does not inhibit EBNA1 transcription in HONE1/
Akata, HK1/Akata and B95-8 cells (Fig. 1d).
To further examine whether PES decreased the
expression of EBNA1 via the proteasome pathway,
HONE1/Akata, CNE1 and HeLa cells were transfected
with the pSG5-EBNA1 and treated with PES in the
absence or presence of the proteosomal inhibitor
MG132. The results showed that PES decreased the
expression of endogenous and exogenous EBNA1 to a similar
degree in the absence or presence of MG-132 (Fig. 1f, g).
In addition, HONE1/Akata and B95-8 cells were treated
with PES in the absence or presence of cycloheximide
(CHX) (50 ?g/ml). As shown in Fig. 1h, CHX did not
attenuate the effect of PES on endogenous EBNA1 protein
level. These results suggested that PES decreases EBNA1
expression independent of effects on EBNA1
transcription or proteasomal degradation.
The glycine?alanine repeats (GAr) domain is not required
for inhibition of EBNA1 expression by PES
EBNA1 contains a central GAr domain that inhibits the
translation of EBNA1 mRNA and play a key role in the
mechanism of immune evasion. To determine if the GAr
domain is required for the inhibition effect of PES on
EBNA1 expression, we compared the effect of PES on the
full-length EBNA1 protein or the mutant EBNA1 lacking
of the GAr domain (EBNA1?GA) protein. HONE1/
Akata, CNE1, and HeLa cells were transfected with the
pSG5-EBNA1 or pSG5-EBNA1?GA, followed by
treatment with PES. These results showed that PES had similar
inhibition effects on the expression of transfected
pSG5EBNA1 and pSG5-EBNA1?GA (Fig. 1e) and MG-132 did
not attenuate effect of PES on expression of EBNA1?GA
protein (Fig. 1i). These results suggested that the GAr
domain is not required for the effect of PES on EBNA1.
PES reduces replication of EBV in HONE1/Akata and
To examine if PES affects EBV replication, HONE1/
Akata and B95-8 cells were treated by TPA (40 or 20 ng/
ml) and NaB (3 mM) to induce the lytic viral replication,
followed by treatment with PES. The intracellular viral
genomic DNA was extracted and the amounts of
intracellular viral DNA were determined by RT-PCR.
Acyclovir (ACV), a drug effectively reducing production of
EBV, was used as a positive drug control32. The results
shown that ACV and PES significantly reduced the
amounts of intracellular viral genomic DNA in induced
HONE1/Akata and B95-8 cells (Fig. 2a, b). EBV
transcription factors Zta, encoded by the viral immediate early
(IE) gene BZLF1, is a lytic switch protein that is necessary
for EBV reactivation and lytic DNA replication33?35. As
shown in Fig. 2c, d, PES reduced expression of EBNA1,
Zta, XIAP and c-IAP1 in induced HONE1/Akata and
B95-8 cells by a dose-dependent manner, but PES did not
affect the expression of Hsp70. Meanwhile, The EBV
Akata genome DNA in HONE1/Akata cells was tagged
with GFP gene, and these cells could be visualized by flow
cytometer analysis. The increase in the intensity of GFP
fluorescence corresponds to the increasing intracellular
viral genome copy numbers. HONE1/Akata cells treated
with induction and PES and the untreated HONE1 cells
were collected and assessed by flow cytometer to analyze
the intensity of GFP fluorescence. The intensity of GFP
fluorescence in HONE1/Akata cells was
dosedependently reduced after PES treatment (Fig. 2e, f).
These results indicated that PES reduces EBV intracellular
DNA replication in HONE1/Akata and B95-8 cells.
PES inhibits proliferation and migration of EBV-positive cells
To determine whether PES affects cell viability of
EBVpositive cells, HONE1/Akata, HK1/Akata, and B95-8 cells,
together with EBV-negative HK2 cells were treated with
vehicle drug control (0.006% DMSO) or a series of
increasing concentrations of PES for 24, 48, and 72 h. As
shown in Fig. 3a?d, PES inhibited the cell viability of
HONE1/Akata, HK1/Akata, and B95-8 cells in a
doseand time-dependent manner, but showed a slight
proliferation inhibition in HK2 cells. In addition,
overexpression of Hsp70 reduced the inhibitory effect of PES
on cell proliferation, but silencing of Hsp70 increased the
inhibitory effect of PES on cell proliferation in HONE1/
Akata and HK1/Akata cells (Fig. 3e?h). To determine the
long-term effect of PES on cell proliferation, HONE1/
Akata and HK1/Akata cells were treated with vehicle
control or PES and cultured for another 15 days. As
shown in Fig. 3i, 40 ?M PES reduced the levels of colony
formation to 10?20% of the levels in HONE1/Akata and
HK1/Akata cells treated with vehicle drug control. The
results suggested that PES inhibits proliferation of
EBVpositive cells efficiently. In order to research further on
whether PES affects the migration capability of
EBVpositive cells, HONE1/Akata and HK1/Akata cells were
treated with the linear scratch wounds, followed by the
treatment of vehicle control or the increasing
concentrations of PES for 24 and 48 h, respectively. As shown
in Fig. 3j?m, PES indeed affects the migration of
PES induces cell cycle arrest and apoptosis in EBV-positive cells
To determine if PES affects cell cycle, HONE1/Akata,
HK1/Akata, and B95-8 cells were treated with vehicle
control or PES for 24 h. The results showed that PES
arrests cell cycles in G2 phase effectively (Fig. 4a). The
ionizing radiation (IR) is one of the most common
therapeutic agents to treat EBV-associated malignancies36.
Meanwhile, it is reported that Hsp70 inhibitors PES could
induce G2 arrest in cancer cells37. In our study, G2 arrest
induced by PES can increase the sensitivity of EBV cells to
radiotherapy (Fig. 4c, d). Then we examined if PES
induces EBV-positive cell apoptosis. As shown in Fig. 4b,
PES resulted in significant cell apoptosis in HONE1/
Akata, HK1/Akata, and B95-8 cells. The western blotting
results also showed that PES can increase the expressions
of cleaved caspase-3 and decrease the expression of Akt,
which indicates that PES can inhibit the proliferation and
promote the apoptosis of EBV positive cells (Fig. 4e).
Autophagy includes microautophagy, macroautophagy,
and chaperone-mediated autophagy (CMA). Hsp70, as an
important regulator of apoptotic signaling pathways, plays
a key role during CMA38,39. To determine if PES induces
apoptosis by inhibiting autophagy, we examined
expression of autophagy markers p62, LC3A/B, and the
lysosomal cysteine peptidase cathepsin D. PES caused
accumulation of p62 and LC3A/B-II and significantly
reduced expression of active-cathepsin D in HONE1/
Akata and HK1/Akata cells (Fig. 4e). Immunofluorescence
assays results showed that cathepsin D is located in the
lysosomes and staining of cathepsin D indicated the
presence of punctate structures in HONE1/Akata and
HK1/Akata cells without PES treatment. After PES
treatment, the staining became diffuse, indicating that
cathepsin D is relocated into the cytosol, which is
consistent with the results of Western blotting (Fig. 4f). These
results showed that PES induces apoptosis by inhibiting
autophagy in HONE1/Akata and HK1/Akata cells.
However, there were no significant changes in the expressions
of LC3A/B, p62, and cathepsin D in B95-8 cells. It is
reported that expression of LMP1 in B95-8 cells could
induce programmed cell death by a way depending on
activation of NF-?B pathway, whereas LMP1 in
EBVpositive NPC cells, including HONE1/Akata and HK1/
Akata cells, does not induce cell death40. Our results
showed that PES treatment causes increased expressions
of LMP1 in HONE1/Akata, HK1/Akata and B95-8 cells
(Fig. 1a). The increased expression of LMP1 may be the
most important factor leading to apoptosis of B95-8 cells.
It was shown that SC-514, which inhibits NF-?B
activation, attenuates the apoptosis induced by PES in B95-8
cells (Fig. 4g). The results showed that PES increases
HeLa cells were
HeLa cells were
with EBNA1 and
HeLa cells were
induced by TPA
expression of LMP1, which may induce apoptosis in
B958 cells by activating caspase through NF-?B pathway.
Hsp70 increases the expression level of EBNA1, but PES inhibits this effect on a dose-dependent manner
To investigate the effect of Hsp70 on EBNA1
expression, HONE1/Akata cells were transfected with
pEF-FlagHsp70 or Hsp70 siRNA, followed by treatment with PES
(20 or 40 ?M). The results showed that over-expression of
Hsp70 increases the expressions of endogenous EBNA1,
XIAP, and c-IAP1, but PES inhibits this effect on a
dosedependent manner (Fig. 5a). Meanwhile, knock-down of
Hsp70 decreased the expressions of endogenous EBNA1,
XIAP, and c-IAP1, but PES increased this effect (Fig. 5b).
In addition, we determined if the GAr domain is required
for the change of EBNA1 expression up-regulated by
Hsp70. The results suggested that the changes of EBNA1
expression induced by Hsp70 are not associated with the
GAr domain in HONE1/Akata and HeLa cells (Fig. 5c, d).
Hsp70 promotes the lytic replication of EBV, but PES inhibits this effect on a dose-dependent manner
To further determine whether Hsp70 affects the lytic
replication of EBV, HONE1/Akata cells were induced by
TPA and NaB, and then transfected with pEF-Flag-Hsp70
or Hsp70 siRNA, followed by treatment with PES. The
copy numbers of the intracellular viral DNA and the
expression of EBNA1 were increased in the induced
HONE1/Akata cells transfected with Hsp70, but PES
inhibited this effect on a dose-dependent manner (Fig. 5e,
f). In addition, the copy numbers of the intracellular viral
DNA and the expression of EBNA1 in the induced
HONE1/Akata cells transfected with Hsp70 siRNA were
decreased, but PES increased this effect on a
dosedependent manner (Fig. 5g, h). These results indicated
that Hsp70 promotes replication of EBV, but PES inhibits
this effect on a dose-dependent manner in HONE1/Akata
EBNA1 interacts with Hsp70, but PES interferes this interaction
The results of co-immunprecipitation assays showed
that EBNA1 interacts with Hsp70 but PES interferes this
interaction (Fig. 6a, b). To further confirm the cellular
localization of EBNA1 and Hsp70, immunofluorescence
assays were performed. The results showed that a strong
signal from EBNA1 overlapped with the Hsp70 signal in
HeLa cells (Fig. 6c?e). Meanwhile, co-location of EBNA1
and Hsp70 was also found in induced HONE1/Akata cells
In vivo effects of PES on BALB/c nude mice inoculated with
Since the above-mentioned results showed the effects
of PES on cells in vitro, the possible effects of PES
in vivo were also investigated. To study if PES influences
the growth of the EBV-positive NPC in BALB/c nude
mice, 1 ? 107 HONE1/Akata cells were subcutaneously
inoculated into the armpit of the mice. After seven
days, the mice were injected intraperitoneally with a
dose of 8 mg/kg PES or control PBS per day for
consecutive five days and then euthanized. The dose of PES
in vivo in this study has been reported to be safe41,42.
PES treatment had no significant effect on the body
weights of mice (Fig. 7a). The tumor weights and
volumes of the PES-treated group were significantly
smaller than the PBS-treated group (Fig. 7b, c).
Representative images of mice and tumors in above mice are
shown in Fig. 7d. Western blotting results of the tumor
tissues indicated that PES significantly down-regulates
the expression of EBNA1, XIAP, and c-IAP1, whereas
Hsp70 expression is not reduced (Fig. 7e). In addition,
the intensity of GFP fluorescence in tumors with PBS
treatment were stronger than that in tumors with PES
treatment (Fig. 7f, g). The results of H&E staining
showed that PES has no significant negative effect on
mice (Fig. 7h). Immunohistochemical staining showed
that the number of EBNA1-positive cells in the tumor
tissues treated with PES were decreased (Fig. 7i). These
results suggested that, in vivo, PES significantly inhibits
the growth of tumors induced by EBV-positive HONE1/
It has been reported that EBV infection is significantly
associated with increased risk and poor prognosis of
EBVrelated diseases. EBNA1 is essential for EBV episome
maintenance, transcription and replication, which are
mediated by EBNA1 binding to homologous OriP DNA12.
This study has shown that the Hsp70 inhibitor PES
effectively inhibited proliferation of EBV-positive cells
in vitro and in vivo. The PES-induced G2 arrest could
increase sensitivity of EBV cells to radiotherapy. PES
induces apoptosis by inhibiting autophagy in HONE1/
Akata and HK1/Akata cells. However, PES increases the
expression of LMP1, which may induce apoptosis in
B958 cells by activating caspase through NF-?B pathway.
Meanwhile, PES inhibited the expression of EBNA1 in
various cell lines. But these effects were not related with
EBNA1 transcription and proteasomal degradation.
Furthermore, the GAr domain was also not necessary for
inhibition of EBNA1 expression by PES. Our findings
suggested that PES down-regulates expression of EBNA1
by a mechanism related with Hsp70.
Our study has shown that PES reduced replication of
EBV in HONE1/Akata and B95-8 cells. The inhibition of
Hsp70 significantly reduced the viral protein synthesis
and virus replication, and PES increased this effect on a
dose-dependent manner. On the contrary, the
overexpression of Hsp70 induced the viral protein synthesis
and virus replication, but PES inhibited this effect on a
dose-dependent manner. The results of EBV infected cells
treated with Hsp70 inhibitor PES in this study
demonstrated that Hsp70 activity was required for efficient
production of EBV, which is consistent with a previous
study43, suggesting that Hsp70 could be considered as a
positive cellular factor for EBV infection.
There is growing evidence that Hsp70 plays essential roles
in replication of many viruses, such as ?-herpesvirus
HSV144, ?-herpesvirus HCMV45 and ? -herpesvirus KSHV46,
suggesting that Hsp70 may be playing important roles in
EBV lytic infection. EBV encodes eight proteins in latent
stage, including the nuclear proteins EBNA-1, -2, -3A, -3B,
-3C, -5, and the membrane proteins LMP-1, -2A, and -2B.
The EBNA genes belong to the same transcription unit and
the different mRNAs are generated by alternative splicing
from a large primary transcript. EBNA3A was reported to
not only interact with the chaperones Hsp70 and the
cochaperones Hsp40 but also up-regulate their expression
levels47. The nucleolus localization of Hsp70 was enhanced
with the presence of EBNA-548. It has been reported that
LANA1 in KSHV can interact with Hsp7049. Although the
full sequences of LANA1 and EBNA1 do not have
similarities, they have similar structures and functions. Therefore,
we speculated that there may also be some correlation
between Hsp70 and EBNA1. Through various experiments,
our study determined that Hsp70 interacts with EBNA1 to
regulate EBV protein synthesis and viral replication, and
that PES inhibits this interaction. It is reported that Hsp70
translocates from cytoplasm to nucleoplasm in response to
stressful treatment, such as heavy metals, heat shock,
ultraviolet radiation and viral infections50,51. In our
experiments, most of transfected Hsp70 co-located with EBNA1
in nucleus of HeLa cells. In HONE1/Akata cells induced by
TPA and NaB, endogenous Hsp70 also co-located with
EBNA1 in the nuclear, which may be related to the stimuli
of EBV. The interaction between Hsp70 and EBNA1 may
explain the inhibitory effect of PES on EBV replication.
As drug resistance is a major obstacle to antiretroviral
therapy, the effectiveness of antiviral drugs is thus severely
limited52. Fortunately, drug-resistance did not emerge
when Hsps inhibitors were used to block numerous
viruses replication, suggesting that the Hsps inhibitors
might become attractive candidates for cure of
virusescaused human disease53. Previous studies have shown that
Hsp70 knockout in mice did not cause pathological
changes in animals, suggesting that reducing Hsp70 levels
in cells appears to be relatively safe54,55. In our study, after
treatment of PES, the organs of the BALB/c nude mice
showed no abnormal lesions. Meanwhile, PES
significantly inhibited the proliferation of HONE1/Akata
cell-induced xenograft tumors in BALB/c nude mice and
decreased the expression of EBNA1 in these tumors.
Notably, these results highlight the therapeutic potential
of PES for the treatment of EBV-related diseases.
Altogether, our study reveals the potential of the novel
Hsp70 inhibitor PES to inhibit EBV replication and
growth of EBV-associated tumors. Therefore, Hsp70
inhibitor PES may provide a novel therapeutic approach
for the treatment of EBV-associated malignancies 46.
Materials and methods
Cell lines, reagents, and antibodies
EBV-positive cells HONE1/Akata and HK1/Akata were
generous gifts given by Prof. S.W. Tsao at the University
of Hong Kong in China. These two cell lines were made
by introducing EBV Akata genome DNA that contains
GFP gene into NPC cell lines HONE1 and HK1 cells.
EBV-positive B lymphoma cell line B95-8 and
EBVnegative NPC cell line CNE1 were kind gifts from Prof. Y.
Cao (Central South University, Changsha, China).
EBVnegative NPC cell line HONE1 was purchased from the
American Type Culture Collection (ATCC; VA, USA).
EBV-negative cell line HK2, a human renal tubular
epithelial cell, was kindly provided by Professor L. Zheng
(Wuhan University, Wuhan, China). HeLa and 293T cells
were kindly provided by Professor H. Li (Wuhan
University, Wuhan, China). HONE1, B95-8, and CNE1 cells
were cultured in RPMI 1640 medium (Hyclone, USA)
containing 10% fetal bovine serum (FBS; Gibco-BRL,
Gaithersburg, MD, USA). HeLa and 293T cells were
maintained in DMEM medium (Hyclone, USA)
containing 10% FBS. For maintaining the recombinant
EBV genomes, HONE1/Akata and HK1/Akata cells were
cultured in RPMI 1640 medium containing 10% FBS and
0.4 ?g/ml Geneticin (G418). HK2 cells were maintained in
DME/F-12 containing 10% FBS and hEGF
(SigmaAldrich, St. Louis, MO, USA).
PES (Merck Millipore, MA, USA), MG-132 (Merck
Millipore, MA, USA), cycloheximide (CHX; Sigma-Aldrich, St.
Louis, MO, USA), 12-O-tetradecanoylphorbol-13-acetate
(TPA; Sigma-Aldrich, St. Louis, MO, USA), and acyclovir
(ACV; Selleck Chemicals, Shanghai, China), SC-514
(SigmaAldrich, St. Louis, MO, USA) were dissolved in
dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA).
Sodium butyrate (NaB; Sigma-Aldrich, St. Louis, MO, USA)
was dissolved in PBS.
The antibodies used in this study were given as follows:
anti-caspase-3 (#9668), anti-cleaved caspase-3 (#9664),
anti-Akt (#4685), anti-XIAP (#14334), anti-c-IAP1
(#7065), anti-cathepsin D (#2284), anti-LC3A/B (#4108),
anti-p62 (#88588), and anti-GAPDH (#5174) were
purchased from Cell Signaling Technology. Anti-EBNA1
(sc81581) and anti-Zta (sc-53904) were purchased from
Santa Cruz. Anti-LMP1 (ab78113), EBV nuclear antigen
(ab8329), goat anti-mouse IgG (ab6789), and goat
antirabbit IgG (ab6721) were purchased from Abcam.
Anti-Hsp70 (ADI-SPA-810-D) was purchased from Enzo.
Anti-Flag (CFLKT002) was purchased from Beijing
Chunfenglv Biomedical Technology (Co., Ltd., Beijing,
China). Goat anti-mouse Alexa Fluor 555 (A21422), Goat
anti-rabbit Alexa Fluor 555 (A21428) and goat anti-rabbit
FITC (F-2765) were purchased from Invitrogen. Goat
anti-mouse DyLight 405 (A23110) was purchased from
Cell viability and colony formation assay
Cell viability assays were performed according to the
instructions, which was described previously56. As for
colony formation assay, cells were digested into cells
suspension with trypsin solution. A total of 4 ml of single cell
suspension was seeded onto 6 cm plates at a density of
200 cells/ml. After adherence, cells were treated with PES
for 48 h and cultured for another 15 days. Then cells were
stained with glutaraldehyde crystal violet mixture
(SigmaAldrich) for 30 min and washed with tap water carefully.
After the plates with colonies being dried, digital images
Cells were grown to confluence on 6 well plates. The
linear scratch wounds were created using a 10 ?l tip, and
then the cells were incubated with culture medium
containing 2.5% FBS. After that, all the cells were treated with
PES. Finally, images of the cells along the scrape line were
captured using microscope after wounding 0, 24, and 48 h,
respectively. The wound-healing capacity was analyzed by
measuring the changes in the width of the wound gaps
using Image J software.
Flow cytometer analysis
Cell cycle was measured using the Annexin V/PI
apoptosis kit (MultiSciences, China) and cell apoptosis
were measured using the Annexin V-Phycoerythrin (PE)/
7-amino-actinomycin D (7-AAD) apoptosis detection kit
(MultiSciences, China) according to the manufacturer?s
directions. Then cells were immediately analyzed by the
flow cytometer (BD FACSAria III, BD) in the Research
Center for Medicine and Structural Biology, Wuhan
Cells were grown to confluence on 6 well plates. Then
the cells were irradiated at a distance of 100 cm at room
temperature by X-ray produced using a Varian medical
linear accelerator (Zhongnan Hosptial). X-irradiation was
carried out at a dose rate of 2 Gy/min.
The plasmids pSG5 vector, pSG5-EBNA1, and
pSG5EBNA1?GA have been described previously57. The
plasmid pEF-Flag-Hsp70 was constructed by inserting the
Hsp70 sequence into the pEF vectors. The plasmids were
isolated using the plasmid DNA extraction kit (cat no.
CFLKP001-50) purchased from Beijing Chunfenglv
Biomedical Technology (Co., Ltd., Beijing, China). The
primers were given as follows: pEF-Flag-Hsp70, forward,
G-3? and reverse, 5?-GCTCTAGACTAGTTCTTCAAAT
For transfection of DNA, cells were transfected with
plasmids using X-treme GENE HP DNA Transfection
reagent (Roche) according to the manufacturer?s protocol.
For siRNA transfection, cells were transfected with
Hsp70 siRNA and control-siRNA molecules (Ribobio,
China) using Lipofectamine RNAiMAX (Invitrogen). The
siRNA primers were as follows: Hsp70 siRNA forward,
5?AGAAGAAGGUGCUGGACAAdTdT-3? and reverse,
Quantitative real-time PCR
Methods for extracting cDNA and genomic DNA have
been previously described56. Expression level of EBNA1
mRNA and the amounts of intracellular viral genomic
DNA were quantified by the CFX96 real-time PCR
detection system using a SYBR Premix Ex Taq kit
(Takara). The quantitative real-time PCR conditions were:
a 30 s-denaturation step at 95 ?C, followed by 40 cycles of
10 s at 95 ?C, 10 s at 60 ?C and 15 s at 72 ?C. Melting curve
analysis was performed from 65?95 ?C (with 0.5 ?C
increments). All data were normalized to the control gene
encoding GAPDH. The primers were given as follows:
EBNA1 forward, 5?-TCATCATCATCCGGGTCTCC-3?
and EBNA1 reverse, 5?-CCTACAGGGTGGAAAAATG
GC-3?2; GAPDH forward, 5?-ACATCGCTCAGACACC
ATG-3? and GAPDH reverse, 5?-TGTAGTTGAGGTCA
Co-immunoprecipitation assays and western blotting
293T cells (5 ? 106) seeded in 10 cm dishes were
transfected with plasmids for 48 h. The cells were harvested and
lysed by lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM
NaCl, 10 mM NaF, 2 mM EGTA, 1 mM Na3VO4, 0.5%
Triton-X-100, and 2 mM DTT) containing cocktail for 30
min on ice. After centrifugation at 12,000 g for 20 min at
4 ?C, the supernatants were collected and incubated with
the Protein A/G PLUS-Agarose beads and appropriate
primary antibody or IgG isotype control antibody at 4 ?C
overnight. After washing four times, these compounds were
mixed with 2? loading buffer and boiled for 5 min at 100 ?C.
The immune complexes were resolved on 10% SDS?PAGE
gels and subjected to western blotting that was performed
according to the instructions as indicated 56.
HeLa cells were grown in chamber slides and
transfected with plasmids. HONE1/Akata cells grown in
chamber slides were induced by TPA and NaB. These
cells were fixed with 4% paraformaldehyde for 10 min and
permeabilized with 0.2% Triton X-100 for 10 min. Then
cells were incubated with blocking buffer (5% BSA in PBS)
for 30 min at 37 ?C, incubated with appropriate primary
antibodies diluted in diluent buffer (1% BSA in PBS)
overnight at 4 ?C and washed four times with PBST (0.3%
Tween 20 in PBS). Next, the cells were incubated with
appropriate second antibodies diluted in diluent buffer for
1 h at 37 ?C. Finally, the nuclear staining was conducted
with 4, 6- diamidino-2-phenylindole (DAPI) for 5 min.
Confocal images were captured using the Leica confocal
LCS-SP8-STED nanoscopes (Leica, Germany).
The procedures and protocols of animal experiments
were approved by the Medical Ethics Committee of Wuhan
University. BALB/c nude mice were purchased and
maintained in the Animal Experiment Center of Animal
Biosafety Level-III Laboratory of Wuhan University. HONE1/
Akata cells (1 ? 107) were subcutaneously inoculated into
the armpit of the 6-week-old male BALB/c nude mice. After
seven days, when tumors had grown to be palpable, the
mice were injected intraperitoneally with a dose of 8 mg/kg
PES or control PBS per day for consecutive five days. Then
the mice were euthanized and the body weights, tumor
weights and volumes were measured. The GFP intensity of
tumors were assessed by the In-vivo Xtreme Imaging
System (BrukerXtreme BI, USA). Hematoxylin and
eosin (H&E) staining and immunohistochemistry analysis
were performed according to the method described in a
previous study 59.
The data were shown as the mean ? standard deviation
(mean ? SD) from at least three independent experiments.
The statistical significance of the difference between any
two samples was evaluated by Student?s t-test using
GraphPad Prism for Windows version 5.0 (GraphPad
Software, USA). The values of P < 0.05 were considered
This work was supported by the National Natural Science Foundation of China
(no. 31270205), the Advanced Talent Independent Research Program of
Wuhan University (no. 410500011) and the Initiative Research Program of
Wuhan University (no. 410100020).
Conflict of interest
The authors declare that they have no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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