EBNA1-targeted inhibitors: Novel approaches for the treatment of Epstein-Barr virus-associated cancers

Abstract

Epstein-Barr virus (EBV) infects more than 90% of humans worldwide and establishes lifelong latent infection in the hosts. It is closely associated with endemic forms of a wide range of human cancers and directly contributes to the formation of some. Despite its critical role in cancer development, no EBV- or EBV latent protein-targeted therapy is available. The EBV-encoded latent protein, Epstein-Barr nuclear antigen 1 (EBNA1), is expressed in all EBV-associated tumors and acts as the only latent protein in some of these tumors. This versatile protein functions in the maintenance, replication, and segregation of the EBV genome and can therefore serve as an attractive therapeutic target to treat EBV-associated cancers. In the last decades, efforts have been made for designing specific EBNA1 inhibitors to decrease EBNA1 expression or interfere with EBNA1-dependent functions. In this review, we will briefly introduce the salient features of EBNA1, summarize its functional domains, and focus on the recent developments in the identification and design of EBNA1 inhibitors related to various EBNA1 domains as well as discuss their comparative merits.

Full text

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5307
T
Th
he
er
ra
an
no
os
st
ti
ic
cs
s
2018; 8(19): 5307-5319. doi: 10.7150/thno.26823
Review
EBNA1-targeted inhibitors: Novel approaches for the
treatment of Epstein-Barr virus-associated cancers
Lijun Jiang1, Chen Xie1, Hong Lok Lung2, Kwok Wai Lo4, Ga-Lai Law3, Nai-Ki Mak2, and Ka-Leung
Wong1
1. Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China.
2. Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China.
3. Department of Applied Biological and Chemical Technology, Hong Kong Polytechnic University, Hung Hom, Hong Kong SAR, China.
4. Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong SAR, China.
 Corresponding authors: Email: klwong@hkbu.edu.hk; nkmak@hkbu.edu.hk; ga-ai.law@polyu.edu.hk; kwlo@cuhk.edu.hk
© Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Received: 2018.04.22; Accepted: 2018.08.14; Published: 2018.10.22
Abstract
Epstein-Barr virus (EBV) infects more than 90% of humans worldwide and establishes lifelong latent
infection in the hosts. It is closely associated with endemic forms of a wide range of human cancers
and directly contributes to the formation of some. Despite its critical role in cancer development,
no EBV- or EBV latent protein-targeted therapy is available. The EBV-encoded latent protein,
Epstein-Barr nuclear antigen 1 (EBNA1), is expressed in all EBV-associated tumors and acts as the
only latent protein in some of these tumors. This versatile protein functions in the maintenance,
replication, and segregation of the EBV genome and can therefore serve as an attractive therapeutic
target to treat EBV-associated cancers. In the last decades, efforts have been made for designing
specific EBNA1 inhibitors to decrease EBNA1 expression or interfere with EBNA1-dependent
functions. In this review, we will briefly introduce the salient features of EBNA1, summarize its
functional domains, and focus on the recent developments in the identification and design of EBNA1
inhibitors related to various EBNA1 domains as well as discuss their comparative merits.
Key words: EBNA1-targeted inhibitor, fluorescent probe, EBV-associated cancers, EBV, EBNA1
Introduction
Epstein-Barr virus (EBV) has been shown to act
as a carcinogenic cofactor in the development of
several lymphoid and epithelial cancers. Since its
discovery in 1964 [1], EBV was found to have a direct
association with a wide range of human malignancies
and has been conclusively linked to infectious
mononucleosis [2]. As a lymphotropic herpesvirus,
EBV can establish lifelong latent infection in the host
[3]. It preferentially infects B cells and occasionally
epithelial cells (Figure 1A) with distinct entry modes
[4]. The entry of EBV into B cells is typically receptor
mediated [5-8], while the infection of epithelial cells
appears to be mediated through cell-to-cell contact
[9-14]. A novel quick and efficient “in-cell infection”
method is used to infect epithelial cells [15]. It has
been reported that non-muscle myosin heavy chain
IIA facilitates EBV infection to nasopharyngeal
epithelial cells [16]. In some cases, EBV infects
CD4+/CD8+ T cells, nature killer cells, smooth
muscle cells, as well as the monocytes, but with less
infection efficiency [3, 17, 18].
Like all herpesviruses, EBV-infected cells
undergo either lytic or latent growth during which
only lytic phase produces infectious viruses with
concomitant cell lysis (Figure 1A). Depending on the
cell type, EBV-infected cells undergo four different
latency programs (0, I, II, III). In infected B cells,
location and the state of differentiation also determine
the form of latency [19]. Naive resting B cells infected
by EBV enter latency III (also called growth
transcription program) with the expression of all nine
known latent proteins, consisting of six nuclear
Ivyspring
International Publisher

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5308
antigens (EBNA 1/2/3A/3B/3C/ EBNA LP) and
three membrane proteins (LMP 1/2A/2B). The
infected cells exit the resting state to become
proliferating lymphoblasts and convert to
lymphoblastoid cell lines (LCLs) [20-22]. Latency III is
the most-studied latency form as it is expressed when
B cells are infected in vitro. While EBV infection in
childhood is usually asymptomatic, infection after
adolescence frequently causes IM, a type of
EBV-associated non-malignancy where latency III is
expressed. EBV latency III in immunocompromised or
post-transplant individuals is strongly associated
with lymphoproliferative disorders. Similar to
antigen-activated B blasts [23], EBV-infected
lymphoblasts might enter follicles, where some of the
cells receive survival signals and undergo
differentiation in the germinal-center to become
memory B cells. Such signals are believed to drive the
cells to enter latency II (also called default
transcription program), a typical form of latency
observed in nasopharyngeal carcinomas, gastric
carcinomas, and Hodgkin’s lymphoma [24-26]. This
latency is presented with a restricted set of expressed
latent proteins: EBNA1, LMP1, LMP2A and LMP2B
[27-29]. Notably, LMP1 is not expressed in
EBV-associated gastric cancer and the secreted protein
BARF1 is expressed in epithelial cells [30-32]. Finally,
the cells leave the follicles as resting memory B cells,
which express only EBNA1 (latency I, also called
EBNA1 only program) or no latent proteins at all
(latency 0, also called latency program), and circulate
between the peripheral blood and Waldeyer’s ring
[33]. Although latency I and latency 0 are commonly
observed in healthy subjects, latency I is also observed
in Burkitt’s lymphoma. The major diseases associated
with EBV latent infection are summarized in Figure
1B.
Figure 1. Overview of EBV infection and key diseases developing from EBV infection. (A) EBV lytic and latent infection in EBV tropistic lymphocytes and
epithelial cells. (B) Key diseases resulting from different infected cell lines in individuals.

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5309
Figure 2. Overview of EBNA1 functional domains.
As discussed above, EBNA1 is expressed in all
latency programs except latency 0, and is also present
in all EBV-positive tumors and represents the only
latent protein in some of these tumors, such as in
latency I Burkitt’s lymphoma. EBNA1 is involved in
the maintenance of EBV episomes in infected cells
[34-36]. The plasmid replication requires the binding
of EBNA1 to two distinct regions in origin of
replication (oriP) [37], the dyad symmetry (DS) and
the family of repeats (FR) [35, 36]. DS contains four
known EBNA1 recognition sites and is considered the
initiation site of DNA replication [38]. The other
functional region, FR, is a cluster of 20 tandem copies
with each possessing an 18 bp palindromic
EBNA1-binding site. FR primarily functions in mitotic
segregation and transcriptional activation [39, 40]. It
can also regulate DNA replication through blocking
the passage of replication forks. No other latent
protein is required for the replication and segregation
of the viral genome by EBNA1, which is also known
to regulate the transcription of other latent proteins by
interacting with specific viral promoters.
The role of EBNA1 in proliferating EBV-infected
cells has largely been confirmed. Several reports have
shown that specific EBNA1 inhibition, including
dominant-negative EBNA1 [41-43], and down-
regulating of EBNA1 expression by antisense
oligodeoxynucleotide result in growth inhibition
[44-46], thus validating EBNA1 as a therapeutic target
in EBV-infected cells. A recent study employed
computational approaches to systematically
investigate the structural details of the “druggable”
binding sites on the EBNA1 protein, and suggested
the feasibility of EBNA1 as a target for drug discovery
[47]. Given the essential and unique roles of EBNA1 in
EBV-associated diseases, as well as the computational
evidence, it is not surprising that EBNA1 has emerged
as one of the intensely studied viral proteins among
EBV latent proteins and constitutes an attractive but
an elusive target for therapeutic intervention of
cancers associated with EBV latent infection.
This review will briefly summarize EBNA1
functional domains together with the recently
identified functional moieties and describe various
strategies that have been exploited to achieve specific
EBNA1 inhibition. The reported compounds will be
compared, for their growth-inhibition effect and
selectivity as well as the employed cell lines.
Inhibitors with in vivo applicability or
clearly-identified mechanisms will be highlighted.
Overview of EBNA1 functional domains
EBNA1 is the first identified EBV protein and the
only viral protein present in both latent and lytic
phases. It is a well-defined DNA-binding protein that
contains several functional domains (Figure 2).
The core DNA-binding and dimerization
domain (DBD/DD) of EBNA1 is located at the
C-terminus within a.a. 459-607 and is involved in all
EBNA1 functions associated with oriP-binding

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5310
[48-50]. EBNA1 DBD/DD binds oriP at the 18-bp
palindromic site as a homodimer [51]. X-ray
crystallography of DBD/DD in its apo- and
DND-bound form has been described [52, 53].
EBNA1 can stably interact with ubiquitin-
specific protease 7 (USP7/HAUSP), for which the
binding domain was mapped to amino acid residues
442-447 [54, 55]. EBNA1-USP7 binding is not required
for its functions such as replication, segregation, and
transactivation, but it plays an important role in
regulating EBV DNA replication [54]. USP7 is known
to regulate cell proliferation and apoptosis by
interacting with p53 and Mdm2. USP7 can stabilize
p53 by deubiquitination, resulting in p53-mediated
growth repression and apoptosis [56]. EBNA1 can
compete with p53 to bind the N-terminal a.a. 53-328 of
USP7 [57] with a 10-fold stronger binding affinity [58].
Thus, EBNA1 can displace USP7 binding with p53 to
protect the cells from p53-mediated apoptosis,
probably contributing to cell immortalization,
proliferation, and survival following infection with
EBV [55, 59]. Furthermore, EBNA1 can independently
interact with USP7 and casein kinase 2 (CK2) of the
host cells to disrupt the formation of promyelocytic
leukemia (PML) nuclear bodies (NBs) [60-62]. EBNA1
can enhance the association of CK2 with PML
proteins, which, in turn, phosphorylates PML
proteins and triggers their degradation. The binding
of EBNA1 to USP7 has been shown to mediate the loss
of PML. As PML NBs play an important role in p53
activation [63-65], the EBNA1-mediated disruption of
PML NBs provides another mechanism for EBNA1 to
increase the survival of EBV-infected cells in the
development of nasopharyngeal carcinoma and
EBV-associated gastric carcinoma [59, 60, 62].
EBNA1 primarily resides in the nucleus of
EBV-infected cells. The nuclear localization of EBNA1
is regulated through the interaction between a short
EBNA1 sequence (NLS, nucleus localization sequence,
a.a. 379-386) and two importins (nuclear import
adaptor, α1 and α5) [48, 66-68]. EBNA1 K379 and
R380 were found to be necessary for its nuclear
translocation [69]. Also, phosphorylation of EBNA1
S385 increased EBNA1 NLS-importin α1/α5
interaction and stimulated EBNA1 nuclear
transportation [68, 69]. A recently described
S385-phosphorylated EBNA1-importin α1 crystal
structure confirmed that its increased nuclear
transportation was a result of the enhanced binding
between EBNA1 NLS and its minor binding sites on
importin α1 [70].
Two linking regions (LR1 a LR2) are located at
the N-terminal (a.a. 40-89) and central (a.a. 325-379)
regions of EBNA1, each of which contains a
Gly-Arg-rich domain (Gly-Arg, a.a. 40-64, a.a.
325-367) and a unique region (UR1, a.a. 64-89; UR2,
a.a. 367-379). Gly-Arg domains facilitate DNA looping
in vitro [71-73]. The central Gly-Arg domain and UR1
are responsible for transcriptional activation by
EBNA1 [74, 75]. Gly-Arg interacts with several
nucleosome-associated proteins for transactivation by
EBNA1 [54, 76, 77]. The zinc ion is reported to be
required for both transcriptional activation and
self-association at UR1, which is regarded as a second
dimeric interface of EBNA1 [78]. Also, UR1 was
shown to associate with the bromodomain-containing
protein 4 (Brd4) that facilitates transcriptional
activation by EBNA1 [79]. Besides regulating
transactivation, another key feature of central Gly-Arg
domain is tethering EBNA1 (a.a. 325-376) to
chromosomes for segregation of EBV episomes.
Several studies have shown that the segregation by
EBNA1 is achieved through attaching to
EBNA1-binding protein 2 (EBP2) on the mitotic
chromosomes [80-82], and both Gly-Arg regions were
shown to bind several RNAs in vitro [83].
The Gly-Gly-Ala repeat region (a.a. 89-325) can
decrease human leukocyte antigen (HLA) class I
presentation of EBNA1 antigens by blocking
proteasome-dependent degradation of these antigens,
and weakens the T-cell response to the EBV latent
infection [84, 85]. This is supported by the evidence
that targeting EBNA1 for rapid degradation can
enhance CD8+ T cell recognition [86]. Also, the
Gly-Gly-Ala repeat domain negatively regulates
EBNA1 translational efficiency to maintain low
EBNA1 levels for evading the immune system of the
host [87, 88]. The reduction in the translational
efficiency of EBNA1 could be due to the formation of
G-quadruplex in the Gly-Gly-Ala repeat region of the
EBNA1 mRNA [89, 90].
Strategies for blocking EBNA1 expression
or EBNA1-dependent functions
Small-molecule or peptide inhibitors for proteins
or protein-protein interactions have long been
investigated with limited success [91-94]. Though the
EBV viral protein EBNA1 was identified in 1971, the
research was mostly focused on its critical functions
(for reviews: [95-97]), and specific inhibition of
EBNA1 as a potential therapeutic approach was
investigated only in the last decade. Nevertheless, the
growing body of studies of EBNA1 inhibition
demonstrated that EBNA1 is a potential target for
therapeutic intervention (Figure 3).

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5311
Figure 3. Recent developments of EBNA1 inhibitors. The inhibitors are shown in their respective EBNA1 domains.
Figure 4. Chemical structures of Hsp90 inhibitors.
Inhibitors requiring Gly-Gly-Ala repeats
The first series of small-molecule inhibitors for
EBNA1 belong to heat shock protein 90 (Hsp90)
inhibitors reported by Kenney and colleagues in 2009
(Figure 4) [98, 99]. Hsp90 is known to facilitate
folding, stabilization, and some functions of its
associated proteins (also called client proteins). The
Hsp90 inhibitors decreased EBNA1 expression in
Burkitt’s lymphoma and NPC cell lines without
affecting its stability and half-life. The inhibition of
EBNA1 expression was dependent on Gly-Gly-Ala
repeats, because no decrease in the expression was
observed for mutant EBNA1 lacking Gly-Gly-Ala
repeats (EBNA1 ∆GA). Consistent with this
observation, EBNA1 translation was decreased by
Hsp90 inhibitors for full-length EBNA1, but not
EBNA1 ∆GA. Furthermore, Hsp90 inhibitors
exhibited significant growth-inhibition in both
EBV-immortalized LCLs and EBV-induced
lymphoproliferative disease in SCID (severe
combined immunodeficient) mice. The inhibitory
effect resulted from the decreased expression level of

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5312
EBNA1. Thus, the Hsp90 inhibitors investigated in
this study can be potentially used to treat
EBV-associated diseases.
Hsp90 inhibitor (17-DMAG) treatment of
LCL-EBNA1 ∆GA lines caused significant growth
inhibition, while it did not decrease the expression of
EBNA1 ∆GA. Since EBNA1 ∆GA should not affect any
of the essential functions of EBNA1, this observation
does not support the hypothesis that the decreased
EBNA1 expression level contributed to
growth-inhibition by Hsp90 inhibitors. This study
also failed to describe the detailed mechanism by
which Hsp90 inhibitors exhibited significant
inhibition. Based on the recent reports that showed
Hsp90 inhibitors decreased the expression of some
oncogenic Hsp90 clients [98, 100], the authors
hypothesized that EBNA1 was a client protein of
Hsp90, which was disproved in the
immunoprecipitation experiments.
Inhibitors blocking EBNA1-DNA binding
The research related to EBNA1 inhibitors
focused on blocking EBNA1-DNA binding. Inhibitors
have been reported that either competitively bind to
EBNA1 or EBNA1-bound DNA and thus interfere
with EBNA1-DNA binding activity. The X-ray
crystallographic structure of EBNA1 DBD/DD in the
apo- and DNA-bound form [52, 53] also facilitated
identification of EBNA1 inhibitors by using screening
techniques since EBNA1 has no known ortholog gene
in humans.
The first inhibitor series of this kind was
identified by Lieberman and colleagues through
high-throughput in silico virtual screening [101]. This
series identified four small molecules: SC7, SC11,
SC19 and SC27 (Figure 5, upper panel), which, except
for SC27, inhibited EBNA1-DNA binding (IC50 in the
micromolar range). Although all three compounds
could almost completely block EBNA1-mediated
transcription, selective inhibition was only observed
with SC19, as SC7 and SC11 could also
non-specifically reduce the unrelated Zta-mediated
transcription. Furthermore, SC11 and SC19 could also
reduce the EBV genome copy number in the Raji
Burkitt lymphoma cell line
to 10-25%, while SC7
showed no apparent effect.
SC7 and SC19 were
recognized as the two top
candidates and underwent
molecular docking
analysis. The simulation
results could explain their
inhibitory activities (IC50)
against EBNA1-DNA
binding, which were 23
and 49 μM, respectively.
Besides binding to the
DNA-binding sites of the
EBNA1 protein, SC7 was
also predicted to align well
with the EBNA1-bound
DNA sequence, which was
not observed with SC19
and could be due to its
bulky phenyl group
imparting a different
orientation when bound
with EBNA1. These
docking simulation results
of SC7 and SC19 provide
valuable information for
future development of
EBNA1-DNA binding
inhibitors to increase the
drug potency and
selectivity.
Figure 5. Chemical structures of EBNA1-DNA binding inhibitors. Inhibitors in the upper and middle panel are
identified via scrrening approaches that could block EBNA1-
DNA binding. The inhibitor in the lower panel is a known
DNA ligand that competitively binds the EBNA1-bound site on DNA.

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5313
In a separate study, the same group conducted
another high-throughput screening and identified
three more compounds LB2, LB3, and LB7 (Figure 5,
middle panel) [102]. LB7 could selectively inhibit
EBNA1-DNA binding, as reflected by its IC50 values (1
µM for EBNA1 binding and no observable inhibition
for Zta binding), and its inhibitory activity was more
potent than that of SC7 in a parallel comparison (2 µM
for EBNA1 and 237 µM for Zta). In addition, LB7 was
the only new compound that could reduce the EBV
copy number (at 5 μM) but a high dose (100 μM) was
required to inhibit EBNA1-induced transcription
partially. It should also be noticed that the high
concentration used in the transcription repression
assay could cause cell death.
Screening approaches have been widely used for
the development of small-molecule inhibitors and
peptide inhibitors, such as for the identification of
inhibitors against EBNA1-DNA binding [103-105]. It
is well-documented that EBV viral genome loss causes
cell apoptosis [41, 106-108], a phenomenon exhibited
by some of the above-mentioned screened
compounds. However, the effect of these inhibitors on
cell growth was not examined in the previous studies.
Furthermore, as pointed out by the investigators,
these candidates would not likely be clinically
relevant unless significant structural modifications
are made to increase their specificity to the protein
EBNA1.
An alternative strategy to block EBNA1-DNA
binding is to occupy EBNA1 binding sites on viral
DNA, typically the 5-‘TAGCA-3’. A pyrrole-imidazole
series was synthesized and investigated for the ability
to target specifically the EBNA1-binding sequence
and thereby affect EBNA1-dependent biological
functions [109]. Among these DNA ligands, DSE-3
(Figure 5, lower panel) had the best performance in
inhibiting EBNA1-DS interaction. DSE-3 showed
selective, though not significant, growth inhibition in
EBV-positive cells (IC50, ~60 µM in three LCLs and
>80 µM in Raji). DSE-3 could also reduce EBV genome
copy number, suppress the expression of EBNA1,
EBNA2 and LMP2, and prevent EBV-induced
transformation of primary B cells. Despite these
positive outcomes, DSE-3, and other DNA ligands,
could possibly target the genomic DNA of the host
cells, and thus affect the expression of some non-viral
genes facilitated by EBNA1-host genome interaction
[110, 111]. Thus, effects of these DNA ligands on the
host genome remain to be further defined.
Nevertheless, DSE-3 provided an interesting insight
for the blockage of EBNA1-DNA binding.
Inhibitors blocking EBNA1-dependent
episome maintenance or transcription
By employing a screening approach, Kang et al.
identified two small molecules, roscovitine and H20,
against transactivation by EBNA1-oriP interaction
(Figure 6, upper panel) [112, 113]. Because roscovitine
is a known inhibitor of cyclin-dependent kinases
(CDKs), eukaryotic linear motif (ELM) analysis of
EBNA1 was performed that suggested serine 393 as a
putative CDK site. The S393A mutant of EBNA1 was
thereby employed, which confirmed that the
transactivation inhibition by roscovitine depends on
serine 393. Roscovitine could decrease the nuclear
EBNA1 amount and increase cytoplasmic EBNA1,
while EBNA1 nuclear/cytoplasmic distribution in the
S393 mutant was unaffected by roscovitine treatment.
Also, roscovitine could reduce EBV episome DNA
and inhibit cell growth of LCLs. This study illustrated
the role of S393 in nuclear import of EBNA1. In
contrast, a subsequent study showed that mutation of
S393 did not affect EBNA1 nuclear localization and its
functions related to EBV DNA replication or
segregation [114]. Instead, the S393 mutation
abrogated PML NBs disruption by EBNA1 by binding
to CK2, as mentioned previously [114].
The other small-molecule inhibitor, H20,
prevented transactivation by EBNA1 and showed
association with EBNA1. However, it failed to inhibit
EBNA1-DNA binding directly. This observation was
explained by a docking study, which suggested that
the docking site for H20 in EBNA1 differs from the
oriP FR binding pocket. To improve the ability to
block EBNA1-DNA binding, structure-activity
relationship analysis of H20 was conducted, which
suggested H31 (Figure 6, upper panel) [113]. H31
showed inhibition of DNA binding, transactivation,
replication, and EBV episome maintenance by
EBNA1. H31 also exhibited selective inhibition of
EBV-positive cells (10 µM, decreased by ~70%).
Although H31 interfered with EBNA1-DNA binding,
it failed to a show direct binding with EBNA1 DBD in
a subsequent surface plasmon resonance (SPR) assay.
Inhibitors blocking linking regions (LR1 and
LR2)-dependent functions
Since RGG domains are known to bind with
RNA [83], a study using electrophoresis mobility shift
assay (EMSA) was performed to investigate whether
EBNA1 prefers the structured RNA. The analysis
confirmed the preference of G-quadruplex RNA to
EBNA1 [115]. Since the RGG-like motif in LR1 and
LR2 also functions in recruiting origin recognition
complex (ORC) to oriP for viral DNA replication,
several G-quadruplex-interacting molecules were
employed to determine whether interference with

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5314
EBNA1-RNA binding affects ORC recruitment.
EBNA1-ORC2 association was most efficiently
disrupted by BRACO-19 (Figure 6, middle panel),
suggesting a necessary role of EBNA1-RNA binding
for the recruitment of ORC by EBNA1. Thus,
BRACO-19 was selected for further investigation.
Treatment of Raji Burkitt lymphoma cell line with 10
µM BRACO-19 for three days decreased the EBV
genome copy number to ∼75%, while a longer
treatment of 6 days caused growth inhibition in both
Raji and LCLs. It also showed a modest inhibition of
mRNA expression of EBNA2 and EBNA3A (∼20%),
and decreased DNA replication and metaphase
attachment by EBNA1.
Since AT-hooks on LR1 and LR2 (two Gly-Args)
allow binding between EBNA1 and AT-rich DNA,
AT-rich binders may interfere with this binding.
Netropsin (Figure 6, lower panel) can bind to the
minor groove of the AT-rich sequence of dsDNA
[116], and has therefore been studied for its inhibitory
activities on EBV genome replication and
EBV-positive cell growth. Treatment with 10 µM
netropsin could induce ~50% EBV plasmid loss as
compared to the control group showing ~30% loss; 50
µM netropsin caused 53% growth inhibition in
EBV-positive cells versus 31% inhibition in
EBV-negative cells. Therefore, the inhibition by
netropsin failed to show selectivity or efficiency. Also,
whether netropsin could inhibit EBNA1–AT-rich
DNA binding was not studied.
The strategies discussed in this section, as well as
the previously described DNA ligands represent
indirect approaches to inhibit EBNA1 function. Rather
than directly binding to EBNA1, these ligands
competitively interact with components EBNA1
associates with, thereby impairing functions exerted
by EBNA1. These approaches may have limited
applications. Especially, molecules that bind to
EBNA1-binding sequences on viral DNA or RNA
might also affect normal functions of host cells by
binding to host DNA or RNA. From this perspective,
small molecules that directly interact with EBNA1
domains are a preferred choice.
Inhibitors based on truncated peptides from
EBNA1 dimeric interface
Rather than small-molecule inhibitors, several
peptides have been identified by using rational
biochemical screening of the EBNA1 DBD/DD
domain as EBNA1-DNA-binding inhibitors [117].
Three peptides, P83 (a.a. 552-566), P84 (a.a. 556-570),
and P85 (a.a., 560-574), sharing the same a.a. sequence
560-566, showed almost complete blockage of
EBNA1-DNA binding. Also, P85 was found to
strongly associate with EBNA1 DBD/DD, as
indicated by SPR assay, and treatment with P85
caused more than 50% decrease in EBNA1
transcription. Thus, a truncated peptide from EBNA1
DBD/DD was able to interfere with
DNA binding and transcription by
EBNA1. The study that previously
discovered roscovitine also identified
another small molecule EiK1 [112]. Like
P85, EiK1 weakened DNA binding and
transcription by EBNA1; however, it
quickly dissociated from the dimeric
EBNA1 in the SPR assay. The ability of
EiK1 and P85 in interfering EBNA1
dimer formation was also studied via
DSS crosslinker-mediated dimerization
and yeast two-hybrid assays; the
results of these two independent assays
suggest that EiK1 seems to disrupt the
self-association of the EBNA1 protein.
In addition, whether the inhibition of
EBNA1 function by P85/EiK1 can
cause growth-inhibition in
EBV-infected cells remains to be
clarified.
The current pool of EBNA1
inhibitors mainly consists of small
molecules. The study by Kim and
co-workers indicated that EBNA1
DBD/DD is experimentally
Figure 6. Chemical structures of inhibitors that block EBNA1-
oriP transactivation and EBNA1
Gly-Args-dependent functions.

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5315
“druggable” by peptides that can inhibit EBNA1
function [117]. Despite encouraging results, effects of
the peptides on cellular growth have not yet been
studied. Furthermore, the poor water-solubility of the
hydrophobic peptide is still a problem that has to be
dealt with.
Inspired by the above study, JLP2 (Figure 7,
upper panel) was designed to increase cell
permeability and was fluorescently labeled for
molecular tracking [118]. The conjugates, containing a
peptide inhibitor and a water-soluble fluorophore,
ensured selective cellular uptake and growth-
inhibition in EBV-positive cells. The EBV-positive
nasopharyngeal carcinoma cell line C666-1 treated
with 20 µM JLP2 showed a ∼50% decrease in cell
viability, while no obvious inhibition was observed in
the EBV-negative HeLa cells. The higher cytotoxicity
of peptide conjugate compared to the unconjugated
peptide may be due to the increased cell permeability,
as indicated by confocal imaging and cellular uptake
results. Molecular docking suggested JLP2 had a
stronger EBNA1 binding than either JLP1 or P2.
Luminescence titration analysis confirmed the
stronger binding by JLP2, where a 1.5-fold emission
enhancement was only observed for JLP2 upon
addition of EBNA1.
The design of JLP2 represents a step forward, but
its profile needs to be optimized including its
non-specific cellular localization and its
growth-inhibition of Burkitt’s lymphoma line. We
therefore designed a new series of probes,
L2P2/L2P3/L2P4 [119-121], by incorporating an NLS
(RrRK) moiety in the probe skeleton, which enabled
nuclear localization and targeted nuclear EBNA1
(Figure 7, lower panel) [48, 122]. Molecular dynamics
simulations suggested an unexpected role of RrRK,
which formed salt bridges among several residues in
the aspartate-rich tail of EBNA1 and thus contributed
to stabilization of the NLS-containing probe–EBNA1
complex. Nuclear localization was demonstrated by
the NLS-containing L2P3 and L2P4, whereas L2P2 only
remained in the cytoplasm. The probe, L2P4,
responded significantly to binding with EBNA1,
exhibiting an 8.8-fold enhancement with a 25 nm
blueshift of its emission because of the introduced
intramolecular charge transfer-characterized
fluorophore. The emission of this kind of fluorophore
is highly solvent-dependent, thus protein-binding
activity causes a change in its fluorescence, with a
more significant change representing stronger
binding activity. Among all tested samples, L2P4
exhibited the strongest binding to EBNA1, qualifying
it as the best inhibitor in EBV-positive cells including
NPC and Burkitt’s lymphoma cell lines. In vivo
growth inhibition by L2P4 in EBV-positive tumors was
also confirmed. Furthermore, cross-linking
dimerization assay results indicate that L2P4 likely
interferes with the self-association of EBNA1 by direct
binding to the dimeric interface. Moreover, the
significant emission enhancement of L2P4 could also
be potentially used to visualize cellular EBNA1. Thus,
by resolving many drawbacks of traditional peptide
inhibitors, L2P4 stands out as a novel solution for
EBV-infected cells.
The key properties of various EBNA1 inhibitors
are summarized and compared in Table 1, including
the time required and the employed cell lines.
Notably, EBNA1 has a long half-life, which is usually
more than 24 h [54, 123], so it requires a relatively long
time for treatment by inhibitors in a viability assay.
Table 1. Comparison of in vitro growth-inhibition by EBNA1 inhibitors
Inhibitor
Conc. (µM)
Time (days)
EBV-positive cell lines
Cell number (cells/100 µL)
Inhibition (%)
Inhibition towards EBV(-) cells (%)
Hsp90 inhibitor – 17-DMAG
0.03
5
LCL1
1×10
4
~100
Not found
LCL2
1×10
4
~100
DNA ligands – DSE3
40
8
LCL-1
Not mentioned
~70
Not found
LCL-2
~70
LCL-3
~70
Raji
~50
roscovitine
5
16
GM3324 LCL
optimal growth density
~85
Not mentioned
H31
10
7
AKATA
EBV(+)
Not mentioned
~78
Not found
6
1022 LCL
~66
BRACO-19
10
6
Raji
2.5×104
~11
Not found
LCL3456
~14
LCL3472
~22
netropsin
50
15
SaII BL
4-2×10
4
~53
~31
JLP2
20
1
C666-1
1×10
4
~51
Not found
L
2
P
4
20
1
C666-1
3×103
~72
Not found
NPC43
~72
Raji
~53

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5316
Figure 7. Chemical structures of inhibitors based on peptides from EBNA1 DBD/DD. Adapted with permission from [118, 119], copyright 2014 The
Royal Society of Chemistry and 2017 Springer Nature.
Conclusions
In the past decades, EBV has drawn attention
due to its substantial contribution in the development
of several lymphoid and epithelial malignancies. The
crucial and unique roles of EBNA1 in maintaining
EBV infection make it an attractive target for
therapeutic intervention of EBV-associated cancers.
Efforts from the past decade in the design or
identification of EBNA1 inhibitors have shown some
progress on several fronts. For example, small-
molecule inhibitors against EBNA1-DNA binding or
peptide-based inhibitors from EBNA1 DBD/DD
confirmed the “druggability” of EBNA1 for the
treatment of EBV-positive cancers. Since inhibitors
affecting EBNA1-DNA binding were mainly
identified by screening approaches, they are unlikely
to be structurally related to EBNA1. Therefore, further
structural modifications of these inhibitors are
required to increase their specificity to EBNA1.
Peptides-based inhibitors possess the advantage of
target specificity but suffer other short-comings
including poor stability, short half-life and
susceptibility to degradation by proteases preventing
their effective delivery to the target tumors.
It is important to acknowledge that current
EBNA1-targeted inhibitors are far from perfect and
not ready for the clinic. Multidisciplinary efforts are
required for designing structurally-relevant
EBNA1-targeted inhibitors. In this respect,
co-immunoprecipitation experiments and X-ray
crystallography analysis could provide critical
information on the structure of EBNA1 inhibitor
complex. Similarly, molecular dynamics simulations
as well as docking studies might afford valuable
insights. Although L2P4 has been observed in tumor
sections by imaging studies, monitoring the whole
human body would be more difficult. It would be
worthwhile to employ real-time and deep-penetrating
imaging modalities, such as positron emission
tomography imaging and magnetic resonance
imaging, to gain a better understanding of the
functions of EBNA1-targeted inhibitors.
Abbreviations
Brd4: bromodomain-containing protein 4; CDKs:
cyclin-dependent kinases; CK2: casein kinase 2;
DBD/DD: DNA binding and dimerization domain;
DS: dyad symmetry; EBNA1: Epstein-Barr nuclear
antigen 1; EBP2: EBNA1 binding protein 2; EBV:

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5317
Epstein-Barr virus; ELM: eukaryotic linear motif;
EMSA: electrophoresis mobility shift assay; FR: family
of repeats; HLA: human leukocyte antigen; Hsps: heat
shock proteins; ITC: intramolecular charge transfer;
LCLs: lymphoblastoid cell lines; LR: linking region;
NBs: nuclear bodies; NLS: nucleus localization
sequence; ORC: origin recognition complex; oriP:
origin of replication; PML: promyelocytic leukemia;
SCID: severe combined immunodeficient; SPR:
surface plasmon resonance; UR: unique region; USP7:
ubiquitin specific protease 7.
Acknowledgements
This review was supported by the Hong Kong
Research Grants Council (HKBU 22301615 and
HKPolyU 153021/18P), The Hong Kong Polytechnic
University - University Research Facility in Life
Sciences (ULS), and Hong Kong Baptist University
(RC-IRMS/1617/1B-CHEM).
Competing Interests
The authors have declared that no competing
interest exists.
References
1. Epstein MA, Achong BG, Barr YM. Virus particles in cultured lymphoblasts
from Burkitt's lymphoma. Lancet. 1964; 1: 702-3.
2. Henle G, Henle W, Diehl V. Relation of Burkitt's tumor-associated herpes-ytpe
virus to infectious mononucleosis. Proc Natl Acad Sci U S A. 1968; 59: 94-101.
3. Fields BN, Knipe DM, Howley PM. Fields virology. Philadelphia: Wolters
Kluwer Health/Lippincott Williams & Wilkins; 2007.
4. Hutt-Fletcher LM. Epstein-Barr virus entry. J Virol. 2007; 81: 7825-32.
5. Fingeroth JD, Weis JJ, Tedder TF, Strominger JL, Biro PA, Fearon DT.
Epstein-Barr virus receptor of human B lymphocytes is the C3d receptor CR2.
Proc Natl Acad Sci U S A. 1984; 81: 4510-4.
6. Nemerow GR, Wolfert R, McNaughton ME, Cooper NR. Identification and
characterization of the Epstein-Barr virus receptor on human B lymphocytes
and its relationship to the C3d complement receptor (CR2). J Virol. 1985; 55:
347-51.
7. Tanner J, Weis J, Fearon D, Whang Y, Kieff E. Epstein-Barr virus gp350/220
binding to the B lymphocyte C3d receptor mediates adsorption, capping, and
endocytosis. Cell. 1987; 50: 203-13.
8. Tanner J, Whang Y, Sample J, Sears A, Kieff E. Soluble gp350/220 and deletion
mutant glycoproteins block Epstein-Barr virus adsorption to lymphocytes. J
Virol. 1988; 62: 4452-64.
9. Young LS, Dawson CW, Brown KW, Rickinson AB. Identification of a human
epithelial cell surface protein sharing an epitope with the C3d/epstein‐barr
virus receptor molecule of B lymphocytes. Int J Cancer. 1989; 43: 786-94.
10. Sixbey JW, Yao QY. Immunoglobulin A-induced shift of Epstein-Barr virus
tissue tropism. Science. 1992; 255: 1578-80.
11. Molesworth SJ, Lake CM, Borza CM, Turk SM, Hutt-Fletcher LM. Epstein-Barr
virus gH is essential for penetration of B cells but also plays a role in
attachment of virus to epithelial cells. J Virol. 2000; 74: 6324-32.
12. Oda T, Imai S, Chiba S, Takada K. Epstein-Barr virus lacking glycoprotein
gp85 cannot infect B cells and epithelial cells. Virology. 2000; 276: 52-8.
13. Tugizov SM, Berline JW, Palefsky JM. Epstein-Barr virus infection of polarized
tongue and nasopharyngeal epithelial cells. Nat Med. 2003; 9: 307-14.
14. Borza CM, Morgan AJ, Turk SM, Hutt-Fletcher LM. Use of gHgL for
attachment of Epstein-Barr virus to epith elial cells compromises infection. J
Virol. 2004; 78: 5007-14.
15. Ni C, Chen Y, Zeng M, Pei R, Du Y, Tang L, et al. In-cell infection: a novel
pathway for Epstein-Barr virus infection mediated by cell-in-cell structures.
Cell Res. 2015; 25: 785-800.
16. Xiong D, Du Y, Wang HB, Zhao B, Zhang H, Li Y, et al. Nonmuscle myosin
heavy chain IIA mediates Epstein-Barr virus infection of nasopharyngeal
epithelial cells. Proc Natl Acad Sci U S A. 2015; 112: 11036-41.
17. Savard M, Belanger C, Tardif M, Gourde P, Flamand L, Gosselin J. Infection of
primary human monocytes by Epstein-Barr virus. J Virol. 2000; 74: 2612-9.
18. Guerreiro-Cacais AO, Li L, Donati D, Bejarano MT, Morgan A , Masucci MG, et
al. Capacity of Epstein-Barr virus to infect monocytes and inhibit their
development into dendritic cells is affected by the cell type supporting virus
replication. J Gen Virol. 2004; 85: 2767-78.
19. Babcock GJ, Hochberg D, Thorley-Lawson AD. The expression pattern of
Epstein-Barr virus latent genes in vivo is dependent upon the differentiation
stage of the infected B cell. Immunity. 2000; 13: 497-506.
20. Aman P, Ehlin-Henriksson B, Klein G. Epstein-Barr virus susceptibility of
normal human B lymphocyte populations. J Exp Med. 1984; 159: 208-20.
21. Thorley-Lawson DA, Mann KP. Early events in Epstein-Barr virus infection
provide a model for B cell activation. J Exp Med. 1985; 162: 45-59.
22. Pope JH, Horne MK, Scott W. Transformation of foetal human keukocytes in
vitro by filtrates of a human leukaemic cell line containing herpes-like virus.
Int J Cancer. 1968; 3: 857-66.
23. Liu YJ, Joshua DE, Williams GT, Smith CA, Gordon J, MacLennan IC.
Mechanism of antigen-driven selection in germinal centres. Nature. 1989; 342:
929-31.
24. Thorley-Lawson DA, Babcock GJ. A model for persistent infection with
Epstein-Barr virus: the stealth virus of human B cells. Life Sci. 1999; 65:
1433-53.
25. Babcock GJ, Thorley-Lawson DA. Tonsillar memory B cells, latently infected
with Epstein–Barr virus, express the restricted pattern of latent genes
previously found only in Epstein–Barr virus-associated tumors. Proc Natl
Acad Sci U S A. 2000; 97: 12250-5.
26. Thorley-Lawson DA. Epstein-Barr virus: exploiting the immune system. Nat
Rev Immunol. 2001; 1: 75-82.
27. Fahraeus R, Fu HL, Ernberg I, Finke J, Rowe M, Klein G, et al. Expression of
Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma. Int J
Cancer. 1988; 42: 329-38.
28. Brooks L, Yao QY, Rickinson AB, Young LS. Epstein-Barr virus latent gene
transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1,
LMP1, and LMP2 transcripts. J Virol. 1992; 66: 2689-97.
29. Deacon EM, Pallesen G, Niedobitek G, Crocker J, Brooks L, Rickinson AB, et
al. Epstein-Barr virus and Hodgkin's disease: transcriptional analysis of virus
latency in the malignant cells. J Exp Med. 1993; 177: 339-49.
30. Imai S, Koizumi S, Sugiura M, Tokunaga M, Uemura Y, Yamamoto N, et al.
Gastric carcinoma: monoclonal epithelial malignant cells expressing
Epstein-Barr virus latent infection protein. Proc Natl Acad Sci U S A. 1994; 91:
9131-5.
31. Sugiura M, Imai S, Tokunaga M, Koizumi S, Uchizawa M, Okamoto K, et al.
Transcriptional analysis of Epstein-Barr virus gene expression in EBV-positive
gastric carcinoma: unique viral latency in the tumour cells. Br J Cancer. 1996;
74: 625-31.
32. zur Hausen A, Brink AA, Craanen ME, Middeldorp JM, Meijer CJ, van den
Brule AJ. Unique transcription pattern of Epstein-Barr virus (EBV) in
EBV-carrying gastric adenocarcinomas: expression of the transforming BARF1
gene. Cancer Res. 2000; 60: 2745-8.
33. Babcock GJ, Decker LL, Freeman RB, Thorley-Lawson DA. Epstein-Barr
virus–infected resting memory B cells, not proliferating lymphoblasts,
accumulate in the peripheral blood of immunosuppressed patients. J Exp Med.
1999; 190: 567-76.
34. Lupton S, Levine AJ. Mapping genetic elements of Epstein-Barr virus that
facilitate extrachromosomal persistence of Epstein-Barr virus-derived
plasmids in human cells. Mol Cell Biol. 1985; 5: 2533-42.
35. Reisman D, Yates J, Sugden B. A putative origin of replication of plasmids
derived from Epstein-Barr virus is composed of two cis-acting components.
Mol Cell Biol. 1985; 5: 1822-32.
36. Rawlins DR, Milman G, Hayward SD, Hayward GS. Sequence-specific DNA
binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites
in the plasmid maintenance region. Cell. 1985; 42: 859-68.
37. Yates J, Warren N, Reisman D, Sugden B. A cis-acting element from the
Epstein-Barr viral genome that permits stable replication of recombinant
plasmids in latently infected cells. Proc Natl Acad Sci U S A. 1984; 81: 3806-10.
38. Gahn TA, Schildkraut CL. The Epstein-Barr virus origin of plasmid
replication, oriP, contains both the initiation and termination sites of DNA
replication. Cell. 1989; 58: 527-35.
39. Reisman D, Sugden B. trans activation of an Epstein-Barr viral transcriptional
enhancer by the Epstein-Barr viral nuclear antigen 1. Mol Cell Biol. 1986; 6:
3838-46.
40. Krysan PJ, Haase SB, Calos MP. Isolation of human sequences that replicate
autonomously in human cells. Mol Cell Biol. 1989; 9: 1026-33.
41. Kennedy G, Komano J, Sugden B. Epste in-Barr virus provides a survival factor
to Burkitt's lymphomas. Proc Natl Acad Sci U S A. 2003; 100: 14269-74.
42. Nasimuzzaman M, Kuroda M, Dohno S, Yamamoto T, Iwatsuki K, Matsuzaki
S, et al. Eradication of Epstein-Barr virus episome and associated inhibition of
infected tumor cell growth by adenovirus vector-mediated transduction of
dominant-negative EBNA1. Mol Ther. 2005; 11: 578-90.
43. Imai S, Kuroda M, Yamashita R, Ishiura Y. Therapeutic inhibition of
Epstein-Barr virus-associated tumor cell growth by dominant-negative
EBNA1. Uirusu. 2005; 55: 239-49.
44. Roth G, Curiel T, Lacy J. Epstein-Barr viral nuclear antigen 1 antisense
oligodeoxynucleotide inhibits proliferation of Epstein-Barr
virus-immortalized B cells. Blood. 1994; 84: 582-7.
45. Yin Q, Flemington EK. siRNAs against the Epstein Barr virus latency
replication factor, EBNA1, inhibit its function and growth of EBV-dependent
tumor cells. Virology. 2006; 346: 385-93.

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5318
46. Hong M, Murai Y, Kutsuna T, Takahashi H, Nomoto K, Cheng CM, et al.
Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference
inhibits proliferation of EBV-positive Burkitt's lymphoma cells. J Cancer Res
Clin Oncol. 2006; 132: 1-8.
47. Gianti E, Messick TE, Lieberman PM, Zauhar RJ. Computational analysis of
EBNA1 "druggability" suggests novel insights for Epstein-Barr virus inhibitor
design. J Comput Aided Mol Des. 2016; 30: 285-303.
48. Ambinder RF, Mullen MA, Chang YN, Hayward GS, Hayward SD. Functional
domains of Epstein-Barr virus nuclear antigen EBNA-1. J Virol. 1991; 65:
1466-78.
49. Chen MR, Middeldorp JM, Hayward SD. Separation of the complex DNA
binding domain of EBNA-1 into DNA recognition and dimerization
subdomains of novel structure. J Virol. 1993; 67: 4875-85.
50. Summers H, Barwell JA, Pfuetzner RA, Edwards AM, Frappier L. Cooperative
assembly of EBNA1 on the Epstein-Barr virus latent origin of replication. J
Virol. 1996; 70: 1228-31.
51. Ambinder RF, Shah WA, Rawlins DR, Hayward GS, Hayward SD. Definition
of the sequence requirements for binding of the EBNA-1 protein to its
palindromic target sites in Epstein-Barr virus DNA. J Virol. 1990; 64: 2369-79.
52. Bochkarev A, Barwell JA, Pfuetzner RA, Furey W, Jr., Edwards AM, Frappier
L. Crystal structure of the DNA-binding domain of the Epstein-Barr virus
origin-binding protein EBNA 1. Cell. 1995; 83: 39-46.
53. Bochkarev A, Barwell JA, Pfuetzner RA, Bochkareva E, Frappier L, Edwards
AM. Crystal structure of the DNA-binding domain of the Epstein-Barr virus
origin-binding protein, EBNA1, bound to DNA. Cell. 1996; 84: 791-800.
54. Holowaty MN, Zeghouf M, Wu H, Tellam J, Athanasopoulos V, Greenblatt J,
et al. Protein profiling with Epstein-Barr nuclear antigen-1 reveals an
interaction with the herpesvirus-associated ubiquitin-specific protease
HAUSP/USP7. J Biol Chem. 2003; 278: 29987-94.
55. Saridakis V, Sheng Y, Sarkari F, Holowaty MN, Shire K, Nguyen T, et al.
Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein-Barr
nuclear antigen 1 implications for EBV-mediated immortalization. Mol Cell.
2005; 18: 25-36.
56. Li M, Chen D, Shiloh A, Luo J, Nikolaev AY, Qin J, et al. Deubiquitination of
p53 by HAUSP is an important pathway for p53 stabilization. Nature. 2002;
416: 648-53.
57. Hu M, Li P, Li M, Li W, Yao T, Wu JW, et al. Crystal structure of a UBP-family
deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde.
Cell. 2002; 111: 1041-54.
58. Holowaty MN, Sheng Y, Nguyen T, Arrowsmith C, Frappier L. Protein
interaction domains of the ubiquitin-specific protease, USP7/HAUSP. J Biol
Chem. 2003; 278: 47753-61.
59. Frappier L. Contributions of Epstein–Barr nuclear antigen 1 (EBNA1) to cell
immortalization and survival. Viruses. 2012; 4: 1537-47.
60. Sivachandran N, Sarkari F, Frappier L. Epstein-Barr nuclear antigen 1
contributes to nasopharyngeal carcinoma through disruption of PML nuclear
bodies. PLoS Path. 2008; 4: e1000170.
61. Sivachandran N, Cao JY, Frappier L. Epstein-Barr virus nuclear antigen 1
Hijacks the host kinase CK2 to disrupt PML nuclear bodies. J Virol. 2010; 84:
11113-23.
62. Sivachandran N, Dawson CW, Young LS, Liu FF, Middeldorp J, Frappier L.
Contributions of the Epstein-Barr virus EBNA1 protein to gastric carcinoma. J
Virol. 2012; 86: 60-8.
63. de Stanchina E, Querido E, Narita M, Davuluri RV, Pandolfi PP, Ferbeyre G, et
al. PML is a direct p53 target that modulates p53 effector functions. Mol Cell.
2004; 13: 523-35.
64. Pearson M, Carbone R, Sebastiani C, Cioce M, Fagioli M, Saito S, et al. PML
regulates p53 acetylation and premature senescence induced by oncogenic
Ras. Nature. 2000; 406: 207-10.
65. Fogal V, Gostissa M, Sandy P, Zacchi P, Sternsdorf T, Jensen K, et al.
Regulation of p53 activity in nuclear bodies by a specific PML isoform. EMBO
J. 2000; 19: 6185-95.
66. Fischer N, Kremmer E, Lautscham G, Mueller-Lantzsch N, Grasser FA.
Epstein-Barr virus nuclear antigen 1 forms a complex with the nuclear
transporter karyopherin alpha2. J Biol Chem. 1997; 272: 3999-4005.
67. Ito S, Ikeda M, Kato N, Matsumoto A, Ishikawa Y, Kumakubo S, et al.
Epstein–Barr virus nuclear antigen-1 binds to nuclear transporter karyopherin
α1/NPI-1 in addition to karyopherin α2/Rch1. Virology. 2000; 266: 110-9.
68. Nardozzi J, Wenta N, Yasuhara N, Vinkemeier U, Cingolani G. Molecular
basis for the recognition of phosphorylated STAT1 by importin alpha5. J Mol
Biol. 2010; 402: 83-100.
69. Kitamura R, Sekimoto T, Ito S, Harada S, Yamagata H, Masai H, et al. Nuclear
import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (importin
α5) is up- and down-regulated by phosphorylation of the nuclear localization
signal for which lys379 and arg380 are essential. J Virol. 2006; 80: 1979-91.
70. Nakada R, Hirano H , Matsuura Y. Structural basis for the regulation of nuclear
import of Epstein-Barr virus nuclear antigen 1 (EBNA1) by phosphorylat ion of
the nuclear localization signal. Biochem Biophys Res Commun. 2017; 484:
113-7.
71. Su W, Middleton T, Sugden B, Echols H. DNA looping between the origin of
replication of Epstein-Barr virus and its enhancer site: stabilization of an origin
complex with Epstein-Barr nuclear antigen 1. Proc Natl Acad Sci U S A. 1991;
88: 10870-4.
72. Frappier L, O'Donnell M. Epstein-Barr nuclear antigen 1 mediates a DNA loop
within the latent replication origin of Epstein-Barr virus. Proc Natl Acad Sci U
S A. 1991; 88: 10875-9.
73. Frappier L, Goldsmith K, Bendell L. Stabilization of the EBNA1 protein on the
Epstein-Barr virus latent origin of DNA replication by a DNA looping
mechanism. J Biol Chem. 1994; 269: 1057-62.
74. Wu H, Kapoor P, Frappier L. Separation of the DNA replication, segregation,
and transcriptional activation functions of Epstein-Barr nuclear antigen 1. J
Virol. 2002; 76: 2480-90.
75. Kennedy G, Sugden B. EBNA-1, a bifunctional transcriptional activator. Mol
Cell Biol. 2003; 23: 6901-8.
76. Wang S, Frappier L. Nucleosome assembly proteins bind to Epstein-Barr virus
nuclear antigen 1 and affect its functions in DNA replication and
transcriptional activation. J Virol. 2009; 83: 11704-14.
77. Malik-Soni N, Frappier L. Nucleophosmin contributes to the transcriptional
activation function of the Epstein-Barr virus EBNA1 protein. J Virol. 2014; 88:
2323-6.
78. Aras S, Singh G, Johnston K, Foster T, Aiyar A. Zinc coordination is required
for and regulates transcription activation by Epstein-Barr nuclear antigen 1.
PLoS Pathog. 2009; 5: e1000469.
79. Lin A, Wang S, Nguyen T, Shire K, Frappier L. The EBNA1 protein of
Epstein-Barr virus functionally interacts with Brd4. J Virol. 2008; 82: 12009-19.
80. Shire K, Ceccarelli DF, Avolio-Hunter TM, Frappier L. EBP2, a human protein
that interacts with sequences of the Epstein-Barr virus nuclear antigen 1
important for plasmid maintenance. J Virol. 1999; 73: 2587-95.
81. Wu H, Ceccarelli DF, Frappier L. The DNA segregation mechanism of
Epstein-Barr virus nuclear antigen 1. EMBO reports. 2000; 1: 140-4.
82. Kanda T, Otter M, Wahl GM. Coupling of mitotic chromosome tethering and
replication competence in Epstein-Barr virus-based plasmids. Mol Cell Biol.
2001; 21: 3576-88.
83. Snudden DK, Hearing J, Smith PR, Grässer FA, Griffin BE. EBNA-1, the major
nuclear antigen of Epstein-Barr virus, resembles 'RGG' RNA binding proteins.
The EMBO Journal. 1994; 13: 4840-7.
84. Levitskaya J, Coram M, Levitsky V, Imreh S, Steigerwald-Mullen PM, Klein G,
et al. Inhibition of antigen processing by the internal repeat region of the
Epstein-Barr virus nuclear antigen-1. Nature. 1995; 375: 685-8.
85. Blake N, Lee S, Redchenko I, Thomas W, Steven N, Leese A, et al. Human
CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the
(Gly-Ala)-containing protein requires exogenous processing. Immunity. 1997;
7: 791-802.
86. Tellam J, Sherritt M, Thomson S, Tellam R, Moss DJ, Burrows SR, et al.
Targeting of EBNA1 for rapid intracellular degradation overrides the
inhibitory effects of the Gly-Ala repeat domain and restores CD8+ T cell
recognition. J Biol Chem. 2001; 276: 33353-60.
87. Yin Y, Manoury B, Fahraeus R. Self-inhibition of synthesis and antigen
presentation by Epstein-Barr virus-encoded EBNA1. Science. 2003; 301: 1371-4.
88. Tellam J, Rist M, Connolly G, Webb N, Fazou C, Wang F, et al. Translation
efficiency of EBNA1 encoded by lymphocryptoviruses influences endogenous
presentation of CD8+ T cell epitopes. Eur J Immunol. 2007; 37: 328-37.
89. Murat P, Zhong J, Lekieffre L, Cowieson NP, Clancy JL, Preiss T, et al.
G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA
translation. Nat Chem Biol. 2014; 10: 358-64.
90. Tellam JT, Lekieffre L, Zhong J, Lynn DJ, Khanna R. Messenger RNA sequence
rather than protein sequence determines the level of self-synthesis and antigen
presentation of the EBV-encoded antigen, EBNA1. PLoS Pathog. 2012; 8:
e1003112.
91. Ma Y, Ai G, Zhang C, Zhao M, Dong X, Han Z, et al. Novel Linear Peptides
with High Affinity to alphavbeta3 Integrin for Precise Tumor Identification.
Theranostics. 2017; 7: 1511-23.
92. Beards F, Jones LE, Charnock J, Forbes K, Harris LK. Placental homing
peptide-microRNA inhibitor conjugates for targeted enhancement of intrinsic
placental growth signaling. Theranostics. 2017; 7: 2940-55.
93. Debnath B, Xu S, Grande F, Garofalo A, Neamati N. Small molecule inhibitors
of CXCR4. Theranostics. 2013; 3: 47-75.
94. Arkin MR, Wells JA. Small-molecule inhibitors of protein-protein interactions:
progressing towards the dream. Nat Rev Drug Discov. 2004; 3: 301-17.
95. Frappier L. EBNA1 and host factors in Epstein-Barr virus latent DNA
replication. Curr Opin Virol. 2012; 2: 733-9.
96. Westhoff Smith D, Sugden B. Potential cellular functions of Epstein-Barr
nuclear antigen 1 (EBNA1) of Epstein-Barr virus. Viruses. 2013; 5: 226-40.
97. Frappier L. Role of EBNA1 in NPC tumourigenesis. Semin Cancer Biol. 2012;
22: 154-61.
98. Sun X, Barlow EA, M a S, Hagemeier SR, Duellman SJ, Burgess RR, et al. Hsp90
inhibitors block outgrowth of EBV-infected malignant c ells in vitro and in vivo
through an EBNA1-dependent mechanism. Proc Natl Acad Sci U S A. 2010;
107: 3146-51.
99. Sun X, Kenney SC. Hsp90 inhibitors: a potential treatment for latent EBV
infection? Cell Cycle (Georgetown, Tex). 2010; 9: 1665-6.
100. Bonvini P, Gastaldi T, Falini B, Rosolen A. Nucleophosmin-anaplastic
lymphoma kinase (NPM-ALK), a novel Hsp90-client tyrosine kinase:
down-regulation of NPM-ALK expression and tyrosine phosphorylation in
ALK(+) CD30(+) lymphoma cells by the Hsp90 antagonist
17-allylamino,17-demethoxygeldanamycin. Cancer Res. 2002; 62: 1559-66.

Theranostics 2018, Vol. 8, Issue 19
http://www.thno.org
5319
101. Li N, Thompson S, Schultz DC, Zhu W, Jiang H, Luo C, et al. Discovery of
selective inhibitors against EBNA1 via high throughput in silico virtual
screening. PLoS One. 2010; 5: e10126.
102. Thompson S, Messick T, Schultz DC, Reichman M, Lieberman PM.
Development of a high-throughput screen for inhibitors of Epstein-Barr virus
EBNA1. J Biomol Screen. 2010; 15: 1107-15.
103. Dietrich U, Durr R, Koch J. Peptides as drugs: from screening to application.
Curr Pharm Biotechnol. 2013; 14: 501-12.
104. Geng L, Wang Z, Jia X, Han Q, Xiang Z, Li D, et al. HER2 targeting peptides
screening and applications in tumor imaging and drug delivery. Theranostics.
2016; 6: 1261-73.
105. Janzen WP. Screening technologies for small molecule discovery: the state of
the art. Chem Biol. 2014; 21: 1162-70.
106. Vereide DT, Sugden B. Lymphomas differ in their dependence on Epstein-Barr
virus. Blood. 2011; 117: 1977-85.
107. Vereide DT, Seto E, Chiu YF, Hayes M, Tagawa T, Grundhoff A, et al.
Epstein-Barr virus maintains lymphomas via its miRNAs. Oncogene. 2014; 33:
1258-64.
108. Kirchmaier AL, Sugden B. Dominant-negative inhibitors of EBNA-1 of
Epstein-Barr virus. J Virol. 1997; 71: 1766-75.
109. Yasuda A, Noguchi K, Minoshima M, Kashiwazaki G, Kanda T, Katayama K,
et al. DNA ligand designed to antagonize EBNA1 represses Epstein-Barr
virus-induced immortalization. Cancer Sci. 2011; 102: 2221-30.
110. Canaan A, Haviv I, Urban AE, Schulz VP, Hartman S, Zhang Z, et al. EBNA1
regulates cellular gene expression by binding cellular promoters. Proc Natl
Acad Sci U S A. 2009; 106: 22421-6.
111. Dresang LR, Vereide DT, Sugden B. Identifying sites bound by Epstein-Barr
virus nuclear antigen 1 (EBNA1) in the human genome: defining a
position-weighted matrix to predict sites bound by EBNA1 in viral genomes. J
Virol. 2009; 83: 2930-40.
112. Kang M-S, Lee EK, Soni V, Lewis TA, Koehler AN, Srinivasan V, et al.
Roscovitine inhibits EBNA1 serine 393 phosphorylation, nuclear localization,
transcription, and episome maintenance. J Virol. 2011; 85: 2859-68.
113. Lee EK, Kim SY, Noh KW, Joo EH, Zhao B, Kieff E, et al. Small molecule
inhibition of Epstein-Barr virus nuclear antigen-1 DNA binding activity
interferes with replication and persistence of the viral genome. Antiviral Res.
2014; 104: 73-83.
114. Cao JY, Shire K, Landry C, Gish GD, Pawson T, Frappier L. Identification of a
novel protein interaction motif in the regulatory subunit of casein kinase 2.
Mol Cell Biol. 2014; 34: 246-58.
115. Norseen J, Johnson FB, Lieberman PM. Role for G-quadruplex RNA binding
by Epstein-Barr virus nuclear antigen 1 in DNA replication and metaphase
chromosome attachment. J Virol. 2009; 83: 10336-46.
116. Chakravorty A, Sugden B. The AT-hook DNA binding ability of the Epstein
Barr virus EBNA1 protein is necessary for the maintenance of viral genomes in
latently infected cells. Virology. 2015; 484: 251-8.
117. Kim SY, Song KA, Kieff E, Kang MS. Small molecule and peptide-mediated
inhibition of Epstein-Barr virus nuclear antigen 1 dimerization. Biochem
Biophys Res Commun. 2012; 424: 251-6.
118. Jiang L, Lui YL, Li H, Chan CF, Lan R, Chan WL, et al. EBNA1-specific
luminescent small molecules for the imaging and inhibition of latent
EBV-infected tumor cells. Chem Commun. 2014; 50: 6517-9.
119. Jiang L, Lan R, Huang T, Chan C-F, Li H, Lear S, et al. EBNA1-targeted probe
for the imaging and growth inhibition of tumours associated with the
Epstein–Barr virus. Nat Biomed Eng. 2017; 1: 0042.
120. Wong K-L, Lung H, Mak N-K, Law GL, Wong WT, Cobb SL. New probe for
Epstein-Barr virus protein EBNA1. Cell Chem Biol. 2017; 24: 647-8.
121. Kosowicz JG, Lee J, Ambinder RF. Cancer: Seeing the ebb of a tumour virus.
Nat Biomed Eng. 2017; 1: 0059.
122. Puckett CA, Barton JK. Targeting a ruthenium complex to the nucleus with
short peptides. Biorg Med Chem. 2010; 18: 3564-9.
123. Tellam J, Connolly G, Green KJ, Miles JJ, Moss DJ, Burrows SR, et al.
Endogenous presentation of CD8+ T cell epitopes from Epstein-Barr
virus-encoded nuclear antigen 1. J Exp Med. 2004; 199: 1421-31.