Antiviral Therapy for Epstein-Barr Virus-Associated Diseases REVIEW ARTICLE Jung-Chung Lin

Transcription

Antiviral Therapy for Epstein-Barr Virus-Associated Diseases REVIEW ARTICLE Jung-Chung Lin
Therapy for EBV infection
REVIEW ARTICLE
Antiviral Therapy for Epstein-Barr Virus-Associated Diseases
Jung-Chung Lin
Department of Microbiology and Institute of Microbiology Immunology and Molecular Medicine, Tzu Chi University, Hualien,
Taiwan
ABSTRACT
A unique feature of Epstein-Barr virus (EBV) infection is its ability to establish latency after infection. Replication of EBV can be
effectively inhibited by available antiviral drugs, but latent EBV infection is unaffected by any of these conventional antiviral agents
regardless of the mode of action. Despite the availability of effective antiviral drugs, treatment for EBV infection remains undeveloped.
This article provides an overview on the virology of EBV infection, the mechanisms of drug action, current status of nucleoside
analogs against EBV replication, and other classes of drug with different target sites, and moves toward establishing a novel approach for treatment of latent EBV infection. (Tzu Chi Med J 2005; 17:1-10)
Key words: EBV, replication, nucleoside analogs, antiviral therapy, latent infection
INTRODUCTION
The Epstein-Barr virus (EBV), one of the eight human herpesviruses, was discovered by Epstein and Barr
in 1964, while researching the cause of a lymphoma that
was the most common tumor afflicting children in certain parts of East Africa. The clinical syndrome described
by Dennis Burkitt suggested that this lymphoma might
be due to a virus.
EBV is one of the most common infective agents of
man. Indeed, around 95% of the total worldwide population carries the virus in a persistent, lifelong infection,
which is completely asymptomatic in the vast majority
of cases. Nevertheless, EBV is involved to a greater or
lesser extent with a plethora of clinical conditions, many
of which are malignant. The EBV is best known as the
causative agent for infectious mononucleosis (IM). Classically EBV is associated with two human malignancies,
endemic Burkitt's lymphoma (eBL) and nasopharyngeal
carcinoma (NPC) [1]. In addition, lymphoma in
immunocompromised patients, peripheral T-cell lym-
phoma (PTLs), oral hairy leukoplakia, AIDS-related
immunoblastic lymhomas, Hodgkin's lymphoma, gastric carcinoma, and AIDS-associated smooth-muscletumors (leiomyomas and leiomyosarcomas) are also associated with EBV infection [1]. Recently, a possible
association with a proportion of breast carcinomas has
been suggested [2].
Gastric carcinoma is the most common cancer in
Japan [3,4] and immunoblastic lymphoma is on the increase due to iatrogenic or HIV-induced immunosuppression [5,6]. Most tumors arise in long-term virus carriers many years after primary EBV infection, reflecting the multistep nature of the oncogenic process and its
culmination in the malignant conversion of a single cell
within the virus-infected pool.
EBV infection can be characterized in three phases:
acute, latent, and reactivated. The peripheral blood and
lymphoid organs are ordinarily the sites of dormancy
for latently EBV-infected lymphocytes. However, if the
host becomes immunosuppressed, the latently infected
cells reactivate and resume cellular proliferation and viral
replication. Symptoms of reactivated EBV infection dif-
Received: February 16, 2004, Revised: April 22, 2004, Accepted: May 4, 2004
Address reprint requests and correspondence to: Dr. Jung-Chung Lin, Institute of Microbiology Immunology and Molecular
Medicine, Tzu Chi University, 701, Section 3, Chung Yang Road, Hualien, Taiwan
Tzu Chi Med J 2005° D 17° D No. 1
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J. C. Lin
fer from those of the primary infection, being associated mainly with malignancies.
The increasing number of diseases that are linked
to EBV infection underlies the long-term importance
either of developing an effective vaccine that can protect against disease or, for the virus-associated
malignancies, of developing novel antiviral agents that
can target the virus-carrying cells.
Virology of EBV infection
The DNA of EBV was first characterized in 1970
as a large, linear, double-stranded molecule with a G +
C content of 59%. The entire DNA sequence (over 172
Kbp) of one such strain (B95-8) was determined. This
feat has facilitated a detailed exploration of the molecular biology of the virus.
The encapsidated EBV virion is a double-stranded
linear DNA of approximately 178 - 190 kb interspersed
with direct repeat elements IR1,2,3,4, and a 500 bp repeat element (TR) found in multiple copies at each end
of the linear genome (Fig. 1). After infection the DNA
circularizes via the terminal repeats to form an extrachromosomal episome (Fig. 2) [7], which is maintained
by the host DNA polymerase [7]. Fused terminal fragments identify the episomal form and can be used as an
indicator of clonality [8]. A sequence within the BamHI
C fragment called ori-P for origin of plasmid replication confers the ability to be maintained as an episome
[9,10]. The trans-acting viral latency protein, EBNA-1,
is required for both initiation and maintenance of replication of EBV episomes by binding to the ori-P
sequence, where it also activates a transcriptional enhancer [11,12]. The episome is the molecular basis for
latent EBV infection and its replication, accomplished
by host DNA polymerase, is not sensitive to antiviral
drugs [13]. In contrast, the replication of linear viral
genome in the virus-productive state is accomplished
by a virally encoded DNA polymerase [7]. When virus
Fig. 1.
2
reactivates there is a transition from exclusive replication of episomes to replication of linear genomes. During latent infection, ori-P is used exclusively to replicate viral genomes. Another origin of replication called
ori-Lyt is used for production of virus [14]. EBV episomes not only serve as template for episomal replication once each cell cycle in the S phase, but also may
serve as templates for circular replicative intermediate
forms and generation of linear genomes [15] in which
case ori-Lyt is used. Thus, latent infection may reactivate and produce virus. Virus-producing cell lines are
mostly latently infected; only 5% - 10% of the population of such lines produce virus, but there is continual
spontaneous transition to the productive state. The transition is induced by many chemical inducers, which trigger the action of immediate-early viral transactivators
[16,17].
Replication of linear viral genomes and production
of virus require virally encoded DNA polymerase (Fig.
3), replication cofactors, and viral proteins used for
encapsidation of the genome. In latently infected people
or cell lines, viral reactivation is triggered by the BZLF1,
a trans-acting immediate-early gene product [18-20].
This key transactivator, a transcription factor that is a
member of the B-Zip family, acts with other immediate-early gene products to activate promoters for early
genes, such as those for the early-antigen-diffuse form
(EA-D) and the EBV DNA polymerase. During latent
infection the early genes are silent. For replication of
EBV six viral genes that serve as replication cofactors
are thought to be essential: BALF5, the DNA
polymerase; BALF2, the single-stranded DNA-binding
protein homolog; BMRF1, the DNA polymerase
processivity factor; BSLF1 and BBLF4, the primase and
helicase homologs and BBLF2/3, a potential homolog
of the third component of the helicase/primase complex.
In addition, a uracil DNA glycosylase homolog may
augment replication [21].
The EBV genome: a physical map of replication-related features. Positions of origins of replication for episomes (ori-P)
and productive infection (ori-Lyt) are shown. The terminal repeated sequences are complementary and may be involved in
a replication step. EBERs are small RNA polymerase III transcripts, probably having a regulatory function and expressed
only in latent infection. EBNA-1 is a latency gene that activates ori-P in trans. BZLF-1 (Zta) is an immediate-early AP-1like gene that triggers reactivation of latent infection. gp340 is a viral envelope glycoprotein that mediates attachment to
the cellular receptor for EBV. In addition to DNA polymerase EBV encodes ribonucleotide reductase and thymidine
kinase enzymes.
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Therapy for EBV infection
Fig. 2. The EBV episome: a physical map. The episome is a
unit-length circularized form of the linear EBV genome found in latently infected cells. Circularization occurs via fusion of the terminal repeat
sequences, which are preserved in characteristic numbers in different viral strains. The origin of replication (ori-P) consists of two cis-acting elements, a
motif of 20 × 30 bp repeats spaced 1000 bp from an
area of dyad symmetry containing four similar repeat units. The motif of repeats functions in vitro as
a transcriptional enhancer whose function is
unknown. DNA replication initiates in dyad and proceeds bidirectionally and asymmetrically, stalling in
the motif of repeats. For ori-P function binding of
the trans-acting latency protein, EBNA-1, to the cis
elements is essential. At least three promoters (Cp,
Wp, Fp) for the EBNA-1 open reading frame and
other EBNAs are used in different cell types and
states.
Fig. 3.
The gene product of BALF5 is a 110 kDa polypeptide that displays core enzymatic activity when translated in vitro and expressed in the baculovirus system
[22-24]. BMRF1 protein (EA-D) copurified with EBV
DNA polymerase from infected cells appears to stabilize enzymatic activity [25] by increasing the processivity
of the holoenzyme [26]. Thus, it may be possible to design analogous mimetic peptides to model specific antiviral drugs that block interaction of EBV DNA polymerase with BMRF1 protein.
An unconventional feature of the processing of the
mRNA for the EBV DNA polymerase may provide a
new target for antiviral therapy. The EBV polymerase
message appears to be polyadenylated without the use
of a canonical polyadenylation signal [27]. Cleavage and
polyadenylation of the 3' end of the polymerase mRNA
is accomplished through the intervention of a protein
induced during the pre-replicative activation cascade.
This induced protein, either cellular or viral, probably
the latter, is thought to act in trans to guide cleavage/
polyadenylation in the absence of a conventional signal
[28]. An EBV pre-early protein BMLF1 gene product
that acts post-transcriptionally is a candidate for this
function [28,29]. Thus, a process thought to be entirely
cellular may be modified by the virus.
The origin of DNA replication used by the EBV
DNA polymerase is ori-Lyt which has been localized to
two sites in the EBV genome in most strains (only 1 in
the deleted B95-8 strain); these are the origins used in
the cytolytic cycle for productive EBV infection [14].
Ori-Lyt has a complex structure and requires viral and
auxillary factors including cellular DNA transcription
factors and a transcriptional enhancer. The BZLF1 gene
The EBV DNA polymerase gene and its control regions. The BALF-5 ORF encodes 5.1 kb polymerase mRNA that is
apparently polyadenylated via a non-canonical polyadenylation signal (UAUAAA); this is the authentic mRNA in most
EBV-producing cell lines and in wild-type virus-infected cells. An alternative polyadenylation site upstream is used in
B95-8 cells to produce a 3.7 kb mRNA because the downstream terminus is deleted in the B95-8 genome; there is no
recognizable polyadenylation signal for this site. Message formation in both cases may involve a virally encoded prereplicative protein. The upstream regulatory region of the promoter for the polymerase gene is TATA-less and contains a
response element (ZRE) for the BZLF-1 gene product near the transcription initiation region. There are at least two cisacting regions (cis-1 and cis-2) required for promoter activity, to which the cellular stimulatory proteins, USF-1 and bZip268 bind; the viral immediate-early proteins also activate these elements. Thus this gene, which is only expressed when
cells are replicating virus, is regulated both transcriptionally at its 5' end and post-transcriptionally at its 3' end.
Tzu Chi Med J 2005° D 17° D No. 1
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J. C. Lin
product also binds directly to ori-Lyt. The product of
this origin is a concatameric DNA molecule. The combination of ori-P, ori-Lyt and the terminal repeats of the
genome may comprise an EBV amplicon [14].
Integrated forms of EBV have been characterized
in two cell lines [30,31]. The EBV DNA has integrated
via the TR resulting in a deletion of cell sequences and a
duplication of cell sequences at each end, similar to a
giant transposon. Integrated copies of the linear genome,
are usually intact but sometimes are ori-P deleted [30].
The significance of EBV integration is unknown since
such forms have not been detected in primary infected
tissues [31].
Mode of action of antiviral drugs
The goal of antiviral chemotherapy is to develop an
agent that selectively inhibits the replication of virus in
infected cells without affecting the metabolic processes
of host cells (for a review see reference [32]). The selection of a suitable antiviral drug for treating virus diseases has been hampered by the fact that most antiviral
compounds are capable of interfering with the molecular processes of host cell functions. Thus, the ideal antiviral drug would interfere only with a virally encoded
process and not with processes in uninfected cells. On
the basis of mode of action antiviral drugs can be divided into six categories: (i) those which interfere with
cellular processes required by the virus for its replication;
(ii) those which selectively bind to, or interfere with,
virus-encoded enzymes and thus inhibit their function;
(iii) those that bind to or incorporate into the virus nucleic
acid and therefore inhibit its expression and function;
(iv) those which prevent the processing of the viral precursor polypeptides; (v) those which interfere with the
virus assembly and inhibit the formation of the virus
progeny; (vi) those which modify the viral proteins on
the surface of the viral envelope and thus prevent the
virus from infecting new cells.
Mechanism of selectivity of antiviral drugs
The mechanism of action of acyclovir against herpes simplex virus (HSV) is the best understood of any
of the antiviral drugs. Thus, an overview of the mechanism of action of acyclovir is illustrated below (Fig. 4).
The selectivity of most anti-herpetic agents depends essentially on two virus-encoded enzymes, thymidine kinase (TK) and DNA polymerase. The virus-encoded TK
is able to phosphorylate not just thymidine (dT) but dU,
dC, thymidylate (dTMP), and a variety of nucleoside
analogs, including acyclovir for instance, that do not
contain a pyrimidine base. Thus, HSV-infected cells
contain much more phosphorylated acyclovir than do
4
Fig. 4. An overview of the mechanism of action of acyclovir.
Acyclovir is first phosphorylated by virus-induced
thymidine kinase to its monophosphate (acyclovirMP), which is then converted to acyclovir-DP and
acyclovir-TP by cellular enzymes. Acyclovir-TP acts
as a substrate and is incorporated into the growing
DNA chain opposite a dC residue. Because there is
no 3'-hydroxyl on acyclovir-TP, the viral DNA polymerase freezes at this step in a "dead-end complex",
leading to apparent inactivation of the enzyme, thereby
preventing the synthesis of viral DNA.
uninfected cells [33]. No mammalian TK phosphorylates acyclovir as efficiently as the HSV TK. Acyclovir
is selectively phosphorylated by HSV-TK [33] and the
resultant acyclo-GMP is then further converted to acycloGDP and acyclo-GTP by cellular kinases [34]. The active form, acyclo-GTP, is a more potent inhibitor of the
viral DNA polymerase than it is of DNA polymerase α,
one of the cellular replicative polymerases. Subsequently, acyclo-GTP acts as a competitive inhibitor with
dGTP; thus, high concentrations of dGTP can reverse
inhibition at this stage. Acyclo-GTP in turn serves as a
substrate for viral DNA polymerase and is incorporated
into the growing DNA chain opposite a dC residue, causing chain termination because of the acyclic nature of
ACV-MP (lack of 3'-OH) and inactivation of the
polymerase; the lack of 3'-OH moiety prevents DNA
chain elongation.
EBV assay systems for drug effects depend upon
the use of two virus-producing cell lines, P3HR-1 and
B95-8. Classically, the assay measures reduction of EBV
genome copy numbers as detected by cRNA-DNA [35,
36] or DNA-DNA hybridization in solution [37].
Alternatively, Southern blot hybridization with an EBV
probe of unique genomic sequence has been used to
measure copy numbers [38]. More recently, the most
precise assay for EBV DNA copy numbers was carried
out by real time quantitative PCR [39]. The total number of genomes measured includes both linear forms and
the minor contribution of episomes (approximately 30
copies per cell) [13,38].
Tzu Chi Med J 2005° D 17° D No. 1
Therapy for EBV infection
Current status of anti-EBV drug studies
Many compounds have been synthesized and reported to have selective inhibitory effects against
herpesviruses, HIV, and other DNA or RNA viruses.
Most of these drugs are nucleoside analogs with primary
targets being virally encoded kinase and DNA
polymerase. This class of drug affects only productive
infection, not latent infection. Currently most of the drugs
that are active against EBV replication are agents that
inhibit replication of other herpesviruses. Anti-HIV
agent, azidothymidine (AZT or zidovudine), is the single
exception; of the eight human herpesviruses this drug
inhibits only the replication of EBV. Studies on in vitro
effects of some promising anti-herpesvirus agents that
are effective against EBV replication are summarized
in Table 1. The potency of antiviral drugs is expressed
by the therapeutic index, which is defined by the ratio
of ID50 (the dose required to inhibit 50% of cell growth)
to ED 50 (the dose required to inhibit 50% of viral
replication) using the in vitro P3HR-1 cell culture system.
For details of each study the reader should refer to the
references cited in Table 1.
Inhibition of EBV replication by glycyrrhizic acid
Glycyrrhizic acid (GL) is an active component of
licorice root (Glycyrrhiza radix), which has long been
Table 1.
Drug
used as a demulcent and elixir in Chinese medicine. GL
has been reported to be active against a variety of viruses including HSV-1, VZV, HCMV, HAV, HBV,
HCV, influenza virus [40-48], and more recently SARSassociated coronavirus [49]. In addition to antiviral
activities, various biological effects of GL, such as antiinflammatory activity [50] and interferon inducibility
[51,52] have also been extensively studied. Clinically,
GL has been used to treat patients with chronic active
hepatitis [53,54].
In view of this broad spectrum of antiviral activity,
we decided to test the effects of GL on EBV DNA
replication. The results clearly indicate that GL is active
against EBV replication [55]. The IC50 values for viral
inhibition and cell growth were 0.04 and 4.8 mM,
respectively. The selectivity index (ratio of IC50 for cell
growth to IC50 for viral DNA synthesis) was 120. GL
had no effect on viral adsorption, nor did it inactivate
EBV particles. Time of addition experiments suggested
that GL interferes with an early step of the EBV replication cycle (possible penetration). Thus, GL represents a
new class of anti-EBV compounds with a mode of action different from that of the nucleoside analogs that
inhibit viral DNA polymerase.
For purposes of comparison, the inhibitory action
of GL against EBV and other viruses such as HIV and
Relative Efficacy of Drugs Active Against EBV Replication in Infected Cell Culture
Antiviral effect ED50 (µM)*
ACV
DHPG
BVdU
BV-araU
V-araU
FMAU
FIAC
FIAU
(S)-DHPA
(S)-HPMPA
PMEA
PMEDAP
(S)-HPMPC
(S)-HPMPDAP
(S)-cHPMPA
AZT
0.3
0.05
0.06
0.26
0.005
0.0065
0.005
0.005
> 100
0.08
1.1
0.16
0.03
2.0
1.5
3
Anticellular effect ID50 (µM)
250
200
360
390
20
1
5
1
80
160
150
155
150
> 50
Therapeutic index ID50/ED50
Reference
830
4000
6000
1500
4000
150
1000
200
1000
1000
5000
78
100
-
13,36,56,57
13,36,56,57
56,57,58,59
59,60
59,60
36,56
36,56
36,56
57.61
57,61
57,61
57,61
57,62
57,62
57,62
57,63
*ED50 was determined by measurement of the reduction of EBV genome copy numbers in the virus-producing P3HR-1 cell line; the 50% inhibitory
dose for cell growth (ID50) was also determined in these cells.
9-(2-hydroxyethoxymethyl)guanine (ACV); E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU); (E)-5-vinyl-1-β-D-arabinofuranosyluracil (V-araU);
(E)-5-(2-bromovinly)-1-β-D-arabinofuranosyluracil (BV-araU); 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodocytosine (FIAC);
1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouridine (FIAU); 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-methyluracil (FMAU);
9-(2-phosphonylmethoxyethyl)adenine (PMEA); (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA];
(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA]; (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diamino-purine [(S)HPMPDAP]; (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine [(S)-HPMPC]; cyclic (S)-HPMPA [(S)-cHPMPA];
9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP); 3'-azido-3'deoxythymidine (AZT)
Tzu Chi Med J 2005° D 17° D No. 1
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J. C. Lin
VZV are summarized in Table 2.
Strategy to cure latent EBV infection
Replication of the EBV can be effectively inhibited
by available antiviral drugs [56-62], but latent EBV infection is unaffected by any of these conventional antiviral agents regardless of the mode of action [32,57].
The difficulty in treating latent forms of viral infection
is a general one that includes not only the herpesviruses
but also all other viruses that produce latent infection.
The EBV genome has two distinct origins of
replication, one, ori-Lyt, used by the viral DNA polymerase in productive infection, and the other, ori-P,
which is the plasmid or episomal origin of replication
[10,14]. The EBV ori-P contains two cis-acting elements,
20 copies of 30 bp family repeats and 4 copies of 15 to
18 bp dyad symmetry, separated by an approximately 1
kb intervening sequence [10]. The motif of family reTable 2.
peated nucleotides is a transcriptional enhancer, and the
dyad array is the site at which episomal DNA replication is initiated [63]. Episomal DNA replication and
maintenance require not only cis-acting elements of the
plasmid ori-P but also a trans-acting function supplied
by a nuclear antigen EBNA-1, which specifically binds
to family repeats and dyad symmetry [12]. In this
connection, cure for latent EBV infection might be accomplished by interference with either synthesis or binding of EBNA-1 to the ori-P. Therefore, a new conceptual approach for treatment of latent EBV infection has
been devised. The strategy focuses on blocking the synthesis of a single viral protein, EBNA-1, which is essential to initiate and maintain replication of EBV episome.
For this purpose, oligodeoxy-nucleotides antisense to
EBNA-1 mRNA were designed and tested in non-virus-producing latently infected cells (Raji) [64,65]. The
most effective oligomers were two sequential 18-mers
Comparison of Relative Potency of GL Against EBV, VZV, and HIV
Viral IC50 (mM)
Virus
EBVb
VZVc
HIVd
0.04
0.71
0.15
Cell IC50 (mM)
4.8
21.3
2.6
Therapeutic Index (cIC50/vIC50)a
120
30
17
a: cIC50 and vIC50 denote cell and viral 50% inhibitory concentration, respectively;
b: The viral IC50 value was obtained by pretreatment of Raji cells with sub-effective dose of GL prior to superinfection. EBV: Epstein-Barr virus;
c: The viral IC50 value was tested in embryonic fibroblasts by plaque assay [41]. VZV: Varicella-zoster virus;
d: The viral IC50 value was determined in MT-4 cells by plaque formation assay [46]. HIV: human immunodeficiency virus
Table 3.
EBV-Associated Diseases and Possible Therapies
Sites and diseases
Epithelial
Nasopharyngeal carcinoma
Hairy leukoplakia
Parotid carcinoma
Lymphoid
Endemic Burkitt's lymphoma
Sporadic Burkitt's lymphoma
B-cell immunoblastic lymphoma
T-cell lymphoma
Hodgkin's disease
Lymphoid/epithelial
Infectious mononucleosis
6
Role of EBV
Essential
Causal
Clonal
Essential
Non-essential, cofactor
Causal
Clonal
Clonal
(Reed-Sternberg cells)
Essential
Possible therapies
Viral DNA pol inhibitor, cytokines, surgery
and radiotherapy
Viral DNA pol inhibitor
Episomal disruption?
Chemotherapy (cyclophosphamide), viral
DNA pol inhibitior
c-myc gene product inhibitor?
Viral DNA pol inhibitor
Episomal disruption?
Episomal disruption?
Viral DNA pol inhibitor, immune modulator,
antibiotics
Tzu Chi Med J 2005° D 17° D No. 1
Therapy for EBV infection
beginning immediately after the AUG of the EBNA-1
ORF. Treatment of Raji cells every 3 days with 40 µM
of the unmodified oligomers over a 23-day period led to
a significant reduction in the expression of EBNA-1.
Concomitant with elimination of synthesis of the EBNA1 protein was a highly significant reduction of EBV episomal copy number by at least 90%. Upon termination
of the treatment, both EBNA-1 protein and episomal
DNA remained below detectable limits longer than 3
weeks. In contrast, the sense oligomers had no inhibitory effects on EBNA-1 synthesis or episomal DNA.
The results indicate that a single viral gene product is
necessary for episomal maintenance. This is the first
successful attempt to block both EBV latent gene expression and episomal replication.
A more recent, conceptually similar strategy resulted
in the growth arrest of EBV-immortalized B cells in cell
culture following administration of an anti-EBNA-1
ribozyme [66]. Ribozymes are catalytic, small RNA species which cleave specific sites within a target mRNA
and can be directed to the particular cleavage site by
complementary base pairing of flanking oligonucleotides. In this case, the ribozyme was delivered and expressed by a recombinant adenovirus, thus being synthesized within the cell (endogenous delivery as compared to exogenous delivery of synthetic oligonucleotides). Both anti-sense and ribozymes have enormous
potential as specific antivirals.
For years, research has focused on effective tools to
specifically down-regulate oncogene overexpression
such as antisense oligonucleotide and ribozyme
strategies. However, there has been only limited success because of the lack of specificity and potency for
these methods. The recent progress of small interfering
RNA (siRNA) techniques has demonstrated the potential to overcome those limitations. The selection of the
targeting sequences of siRNA is less restricted, so the
success rates of producing effective duplexes are higher.
In addition, siRNA is dsRNA, which is more resistant
to nuclease degradation as compared with antisense
oligos and, therefore, have longer therapeutic effects than
the antisense approaches. A recent study directly compared these two techniques and found that siRNA appeared to be quantitatively more efficient with more
durable in cell culture [67].
Despite the advance of these new technologies, a
major remaining problem is delivery and targeting. How
can the agent be specifically and efficiently transported
to the target cells and not "wasted" on other normal cells?
Much effort is being expended in these directions but
much more remains to be accomplished.
Tzu Chi Med J 2005° D 17° D No. 1
CONCLUSIONS
Since EBV interacts in different ways with the cells
that it infects, treatment of EBV infection must ultimately
meet three different objectives: inhibition of active viral
replication; cure of latent viral infection; and interruption of EBV-induced cellular proliferation and transformation (Table 3). It has proven much easier to achieve
the first objective than the others, at least in vitro; however the clinical benefit of inhibiting EBV replication
remains unclear. In humans, treatment of at least two of
these infection states will probably be required for a
definitive outcome. The third infection state, cellular
proliferation and transformation, while it is an inevitable
consequence of EBV infection, rarely has clinical impact except in those that are immunodeficient. These
three infection states are interwoven biologically and
have quite different molecular and cellular mechanisms.
Thus, treatment of EBV infection presents a microcosm
of the issues posed for antiviral drug therapy generally,
including the perplexities of whether treatment of viral
infections that have triggered a neoplastic process will
help to retard or reverse that process (Table 3).
The greatest challenge in antiherpetic therapy may
be that of dealing with latent infection. There are no
drugs, licensed or experimental, regardless of mechanism of action, which have shown any effect on latent
EBV or other herpesvirus infection. This is because EBV
episomes, the probable molecular basis for latent
infection, replicate using host DNA polymerase rather
than viral DNA polymerase. The use of an antisene oligomer or an anti-EBNA-1 ribozyme to block the synthesis of a single selected viral gene product needed to
maintain the EBV episome in latently infected cells appears to be promising, not only for the cure of latent
viral infection, but also for reversal of EBV-associated
diseases. It is also possible that targeting multiple latent
viral genes might mount a synergistic effect that would
prove to be more efficient at curing latent infection. This
new approach represents a revolutionary therapeutic
strategy that directly targets and inhibits gene expression,
laying the foundation for further development of therapies for other herpesvirus latent infections. Thus, a therapeutic strategy using antisense agents, ribozymes, and
siRNA to disrupt episomal replication in combination
with antiviral drugs to inhibit replicative viral DNA
would be necessary to eradicate the virus completely.
These aspects emphasize the need for further basic research into the mechanisms of herpesvirus replication
and latency.
7
J. C. Lin
ACKNOWLEDGEMENTS
I am indebted to Dr. Joseph Pagano for his constant
support during the course of studies, and for the technical assistance of Carolyn Smith, Etsuyo Choi, and
Francoise Besencon. This article is dedicated to Miss
Carolyn Smith, now deceased. This work was supported
by grants from the National Institutes of Health (USA)
(RO1-AI-17205-13), the National Cancer Institute
(USA) (PO1-CA-19104-16), National Cancer Institute
(USA) (RO1-CA-56695-01), and The Burroughs
Wellcome Company. In addition, this work was also
supported in part by grants from the National Science
Council of the Republic of China (NSC 92-3112-B-320001) and by an Institutional Grant (TCMRC 8608) of
the Tzu Chi University.
REFERENCES
1. Rickinson AB, Kieff E: Epstein-Barr Virus. In: Fields BN,
Knipe DM, eds. Fields Virology, 3rd ed., Philadelphia:
Lippincott Raven Publishers, 1996, pp 2397-2446.
2. Bonnet M, Guinebretiere JM, Kremmer E, et al: Detection of Epstein-Barr virus in invasive breast cancers. J
Natl Cancer Inst 1999; 18:1376-1381.
3. Fukayama M, Hayashi Y, Iwasaki Y, et al: Epstein-Barr
virus-associated gastric carcinoma and Epstein-Barr
virus infection of the stomach. Lab Invest 1994; 71:7381.
4. Fukayama M, Chong JM, Kaizaki Y: Epstein-Barr virus
and gastric carcinoma. Gastric Cancer 1998; 1:104-114.
5. Ziegler JL, Drew WL, Miner RC, et al: Outbreak of
Burkitt's-like lymphoma in homosexual men. Lancet
1982; 2:631-633.
6. Cooper DA, Pehrson PO, Pedersen C, et al: The efficacy and safety of zidovudine alone or as cotherapy
with acyclovir for the treatment of patients with AIDS
and AIDS-related complex: A double-blind randomized
trial. European-Australian Collaborative Group. AIDS
1993; 7:197-207.
7. Pagano JS: Gertrude and Werner Henle lecture on viral oncology. From latency to replication: Recent studies of the Epstein-Barr virus. In: Ablashi DV, Faggioni
A, eds. Epstein-Barr Virus and Human Disease, Totowa,
New Jersey: Humana Press, 1991, pp 19-32.
8. Raab-Traub N, Flynn K: The structure of the termini of
the Epstein-Barr virus as a marker of clonal cellular
proliferation. Cell 1986; 47:883-889.
9. 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 USA 1984; 81:
3806-3810.
8
10. 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; 5:1822-1832.
11. Reisman D, Sugden B: Trans activation of an EpsteinBarr viral transcriptional enhancer by the Epstein-Barr
viral nuclear antigen 1. Mol Cell Biol 1986; 6:3838-3846.
12. Rawlins D, 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; 42:859-868.
13. Lin JC, Smith MC, Pagano JS: Prolonged inhibitory effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine
against replication of Epstein-Barr virus. J Virol 1984;
50:50-55.
14. Hammerschmidt W, Sugden B: Identification and characterization of ori-Lyt, a lytic origin of DNA replication of
Epstein-Barr virus. Cell 1988; 55:427-433.
15. Sato H, Takimoto T, Tanaka S, Tanaka J, Raab-Traub
N: Concatameric replication of Epstein-Barr virus: Structure of the termini in virus-producer and newly transformed cell lines. J Virol 1990; 64:5295-5300.
16. Zur Hausen H, O'Neill FJ, Freese UK, Hecker E: Persisting oncogenic herpesvirus induced by the tumor promoter TPA. Nature 1978; 272:373-375.
17. Lin JC, Shaw JE, Smith MC, Pagano JS: Effect of 12-0tetradecanoyl-phorbol-13-acetate on the replication of
Epstein-Barr virus. I. Characterization of viral DNA. Virology 1979; 99:183-187.
18. Countryman J, Miller G: Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a
small cloned subfragment of heterogeneous DNA. Proc
Natl Acad Sci USA 1985; 82:4085-4089.
19. Rooney CM, Rowe DT, Ragot T, Farrell PJ: The spliced
BZLF1 gene of Epstein-Barr virus (EBV) transactivates
an early promoter and induces the virus productive cycle.
J Virol 1989; 63:3109-3116.
20. Kenney S, Kamine J, Holley-Guthrie E, Lin JC, Mar EC,
Pagano JS: The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent
versus productive EBV promoters. J Virol 1989; 63:17291736.
21. Fixman ED, Hayward GS, Hayward D: Transacting requirements for replication of Epstein-Barr virus ori-Lyt.
J Virol 1992; 54:561-568.
22. Lin JC, Sista ND, Besencon F, Kamine J, Pagano JS:
Identification and functional characterization of EpsteinBarrvirus DNA polymerase by in vitro transcription-translation of a cloned gene. J Virol 1991; 65:2728-2731.
23. Lin JC, De BK, Mar EC: Functional characterization of
partially purified Epstein-Barr virus DNA polymerase
expressed in the baculovirus system. Virus Genes 1994;
8:231-241.
24. Tsurumi T, Kobayashi A, Tamai K, Daikoku T, Kurachi
R, Nishiyama Y: Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit. J Virol 1993; 67:4651-4658.
Tzu Chi Med J 2005° D 17° D No. 1
Therapy for EBV infection
25. Li JS, Zhou BS, Dutschman GE, Grill SP, Tan RS, Cheng
YC: Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase
activity. J Virol 1987; 61:2947-2949.
26. Tsurumi T, Daikoku T, Kurachi R, Nishiyama Y: Functional interaction between Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit in
vitro. J Virol 1993; 67:7648-7653.
27. Furnari FB, Adams MD, Pagano JS: Unconventional
processing of the 3' termini of the Epstein-Barr virus
DNA polymerase mRNA. Proc Natl Acad Sci USA 1993;
90:378-382.
28. Furnari FB, Adams MD, Pagano JS: Regulation of the
Epstein-Barr virus DNA polymerase gene. J virol 1993;
66:2837-2845.
29. Kenney S, Kamine J, Holley-Guthrie E, et al: The
Epstein-Barr virus immediate-early gene product,
BMLF1, acts in trans by a post-transcriptional mechanism which is reporter gene dependent. J Virol 1989;
63:3870-3877.
30. Hurley EA, Agger S, McNeil JA, et al: When EpsteinBarr virus persistently infects B-cell lines, it frequently
integrates. J Virol 1991; 65:1245-1254.
31. Gulley ML, Raphael M, Lutz CT, Ross DW, Raab-Traub
N: Epstein-Barr virus integration in human lymphomas
and lymphoid cell lines. Cancer 1992; 70:185-191.
32. Pagano JS: Epstein-Barr virus: Therapy of active and
latent infection. In: Jeffries DJ, Clercq E De, eds. Antiviral Chemotherapy. Chichester, England: John Wiley &
Sons Ltd., 1995, pp 155-195.
33. Elion GB, Furman PA, Fyfe JA, de Miranda P,
Beauchamp L, Schaeffer HJ: Selectivity of action of an
antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine.
Proc Natl Acad Sci USA 1977; 74:5716-5720.
34. Miller WH, Miller RL: Phosphorylation of acyclovir
diphosphate by cellular enzymes. Biochem Pharmacol
1982; 31:3879-3884.
35. Lin JC, Pagano JS: Cellular transformation by the herpesviruses and antiviral drugs. Pharmacol Ther 1985;
28:135-161.
36. Lin JC, Smith MC, Cheng YC, Pagano JS: Epstein-Barr
virus: Inhibition of replication by three new drugs. Science 1983; 221:578-579.
37. Nonoyama M, Pagano JS: Separation of Epstein-Barr
virus DNA from large chromosomal DNA in non-virusproducing cells. Nat New Biol 1972; 238:169-171.
38. Lin JC: Strategies for evaluation of antiviral agents
against Epstein-Barr virus in culture. In: Kinchington D,
Schinazi RF, eds. Methods in Molecular Medicine: Antiviral Chemotherapy Protocols, Totowa, New Jersey:
The Humana Press, 1999, pp 139-150.
39. Lo YM, Chan AT, Chan LY, et al: Molecular prognostication of nasopharyngeal carcinoma by quantitative
analysis of circulating Epstein-Barr virus DNA. Cancer
Res 2000; 60:6878-6881.
40. Pompei R, Flore O, Marccialis MA, Pani A, Loddo B:
Glycyrrhic acid inhibits virus growth and inactivates virus particles. Nature 1979; 281:689-690.
Tzu Chi Med J 2005° D 17° D No. 1
41. Baba M, Shigeta S: Antiviral activity of glycyrrhizin
against varicella-zoster virus in vitro. Antiviral Res 1987;
7:99-107.
42. Numazaki K, Nagata N, Sato T, Chiba S: Effect of
glycyrrhizin, cyclosporin A, and tumor necrosis factor a
on infection of U-937 and MRC-5 cells by human
cytomegalovirus. J Leukoc Biol 1994; 55:24-28.
43. Crance JM, Leveque F, Biziagos E, van Cuyck-Gandre
H, Jouan A, Deloince R: Studies on mechanism of action of glycyrrhizin against hepatitis A virus replication
in vitro. Antiviral Res 1994; 23:63-76.
44. Sato H, Goto W, Yamamura J, et al: Therapeutic basis
of glycyrrhizin on chronic hepatitis B. Antiviral Res 1996;
30:171-177.
45. Arase Y, Ikeda K, Murashima N, et al: The long term
efficacy of glycyrrhizin in chronic hepatitis C patients.
Cancer 1997; 79:1494-1500.
46. Ito M, Nakashima H, Baba M, et al: Mechanism of inhibitory effect of glycyrrhizin on the in vitro infectivity
and cytopathic activity of the human immunodeficiency
virus [HIV(HTLV-III/LAV)]. Antiviral Res 1987; 7:127137.
47. Ito M, Sato A, Hirabayashi K, et al: Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV). Antiviral Res 1988; 10:289298.
48. Utsunomiya T, Kobayashi M, Pollard RB, Suzuki F:
Glycyrrhizin, an active component of licorice roots, reduces morbidity and mortality of mice infected with lethal doses of influenza virus. Antimicrob Agents
Chemother 1997; 41:551-556.
49. Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau
H, Doerr HW: Glycyrrhizin, an active component of licorice roots, and replication of SARS-associated
coronavirus. Lancet 2003; 361:2045-2046.
50. Finney RS, Somers GF: The antiinflammatory activity
of glycyrrhetinic acid and derivatives. J Pharm Pharmacol 1958; 10:613-620.
51. Abe N, Ebina T, Ishida N: Interferon induction by glycyrrhizin and glycyrrhetinic acid in mice. Microbiol
Immunol 1982; 26:535-539.
52. Shinada M, Azuma M, Kawai H, et al: Enhancement of
interferon-gamma production in glycyrrhizin-treated human peripheral lymphocytes in response to convanavalin A and to surface antigen of hepatitis B virus. Proc
Soc Exp Biol Med 1986; 181:205-210.
53. Van Rossum TG, Vulto AG, Hop WC, Brouwer JT,
Niesters HG, Schalm SW: Intravenous glycyrrhizin for
the treatment of chronic hepatitis C: A double-blind,
randomized, placebo-controlled phase I/II trial. J
Gastroenterol Hepatol 1999; 14:1093-1099.
54. Tsubota A, Kumada H, Arase Y, et al: Combined
ursodeoxycholic acid and glycyrrhizin therapy for chronic
hepatitis C virus infection: A randomized controlled trial
in 170 patients. Eur J Gastroenterol Hepatol 1999; 11:
1077-1083.
55. Lin JC: Mechanism of action of glycyrrhizic acid in inhibition of Epstein-Barr virus replication in vitro. Antiviral
Res 2003; 59:41-47.
9
J. C. Lin
56. Lin JC, Smith MC, Pagano JS: Comparative efficacy
and selectivity of some nucleoside analogs against
Epstein-Barr virus. Antimicrob Agents Chemother 1985;
27:971-973.
57. Lin JC: Antiviral therapy for Epstein-Barr virus: The challenge ahead. Recent Res Development Antimicrob
Agents Chemother 1999; 3:191-223.
58. Lin JC, Smith MC, Choi EI, De Clercq E, Verbruggen A,
Pagano JS: Effect of (E)-5-(2-bromovinyl)-2'deoxyuridine on replication of Epstein-Barr virus in human lymphoblastoid cell lines. Antiviral Res 1985;
(Suppl 1):121-126.
59. Lin JC, Machida H: Comparison of two bromovinyl
nucleoside analogs, 1-β-D-arabinofuranosyl-E-5-(2bromovinyl)uracil and E-5-(2-bromovinyl)-2'deoxyuridine, with acyclovir in inhibition of Epstein-Barr
virus replication. Antimicrob Agents Chemother 1988;
32:1068-1072.
60. Lin JC, Reefschlager J, Herrmann G, Pagano JS: Structure-activity relations between (E)-5-(2-bromobinyl) and
5-vinyl-1-β-D-arabinofuranosyluracil (BV-araU, V-araU)
in inhibition of Epstein-Barr virus replication. Antiviral
Res 1992; 17:43-52.
61. Lin JC, De Clercq E, Pagano JS: Novel acyclic adenosine analogs inhibit Epstein-Barr virus replication.
Antimicrob Agents Chemother 1987; 31:1431-1433.
10
62. Lin JC, De Clercq E, Pagano JS: Inhibitory effects of
acyclic nucleoside phosphonate analogs, including (S)1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, on
Epstein-Barr virus replication. Antimicrob Agents
Chemother 1991; 35:2440-2443.
63. 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:
527-535.
64. Lin JC, Raab-Traub N, Pagano JS: Disruption of EpsteinBarr virus episomal DNA maintenance by a specific
oligodeoxyribonucleotide. Antiviral Res 1990; 70 (Suppl
1):76.
65. Pagano JS, Jimenez G, Sung NS, Raab-Traub N, Lin
JC: Epstein-Barr viral latency and cell immortalization
as targets for antisense oligomers. Ann NY Acad Sci
1992; 660:107-116.
66. Huang S, Stupack D, Mathias P, Wang Y, Nemerow G:
Growth arrest of Epstein-Barr virus immortalized B lymphocytes by adenovirus-delivered ribozymes. Proc Natl
Acad Sci USA 1997; 94:8156-8161.
67. Bertrand JR, Pottier M, Vekris A, Opolon P, Maksimenko
A, Malvy C: Comparison of antisense oligonucleotides
and siRNA in cell culture and in vivo. Biochem Biophys
Res Commun 2002; 296:1000-1004.
Tzu Chi Med J 2005° D 17° D No. 1