Rev. Med. Virol. 2005; 15: 149156.

Published online 16 November 2004 in Wiley InterScience (www.interscience.wiley.com).

Reviews in Medical Virology
DOI: 10.1002/rmv.456

REVIEW

Reactivation of Epstein-Barr virus from latency

Wolfgang Amon and Paul J. Farrell*
Ludwig Institute for Cancer Research and Department of Virology, Imperial College Faculty of Medicine,
St Marys Campus, Norfolk Place, London W2 1PG, UK

SUMMARY
The general problem in cancer treatment centres on nding agents that specically affect cancer cells without
damaging normal cells. The differences between cancer cells and normal cells are usually very subtle but about 15% of
all human cancers involve a virus infection, for example the Epstein-Barr virus associated cancers. In these cancers,
every tumour cell carries the virus in a latent infection but the number of normal cells infected is very low. So a
treatment that could somehow cause the elimination of EBV infected cells would be very specic for the cancer in
such cases. One potential approach could involve nding ways to reactivate the latent virus in cancer cells into the
early part of the lytic cycle, impeding cell proliferation, targeting chemotherapeutic agents to the cancer and causing
the cancer cells to become targets for immune surveillance. This review considers the mechanisms by which EBV
reactivation is controlled and discusses possible therapeutic approaches. Copyright # 2004 John Wiley & Sons, Ltd.
Accepted: 13 September 2004

BACKGROUND AND EBV LATENCY

Epstein-Barr virus is a member of the herpesvirus
family and infects more than 90% of the worlds
population. The double-stranded DNA genome
of the virus is about 172 kb in length and encodes
about 85 genes [13]. The life cycle of herpes
viruses is biphasic. Upon primary infection, there
is usually a brief replication of the virus at the site
of infection but this will be conned in an immunocompetent host. The herpes virus then establishes a latent infection in another cell type, in
which the viral genome is maintained indenitely
[1,4]. All herpes viruses share this general strategy
but they realise it in different cell types.
EBV infects mainly B lymphocytes and certain
epithelial cells. Infection normally results in a
latent (non-productive) infection in which the
virus genome is maintained in the nucleus of the
infected cell but no lytic replication of the virus
occurs. In cell culture, lytic replication can be
induced by a variety of treatments that cause
*Corresponding author: Prof. P. J. Farrell, Ludwig Institute for Cancer Research and Department of Virology, Imperial College Faculty of
Medicine, St Marys Campus, Norfolk Place, London W2 1PG, UK.
E-mail: p.farrell@imperial.ac.uk
Abbreviations used
BCR, B cell receptor; BL, Burkitts lymphoma; EBV, Epstein-Barr
virus; LCLs, lymphoblastoid cell lines; PMA, phorbol 12-myristate
13-acetate; PTLD, post-transplantation lymphoproliferative disease;
TGF-, transforming growth factor .

Copyright # 2004 John Wiley & Sons, Ltd.

changes in the cell differentiation state. In vivo,

the virus is transmitted orally via saliva [5] and
infection of B lymphocytes occurs within the lymphoid organs in the oropharynxthe tonsils, adenoids etc of Waldeyers ring. Most primary
infections occur during the rst few years of life
and are asymptomatic but, if primary infection is
delayed until adolescence or adulthood, EBV
infection can cause infectious mononucleosis [6].
The symptoms of infectious mononucleosis are
caused by a large T cell response to a poorly controlled primary infection of B cells by EBV. Once
the EBV infection has been brought under control,
the T cell response (and mononucleosis) subsides
and EBV then persists for the lifetime of the individual by establishing a latent infection in memory
B cellsthe same as a person who was infected
asymptomatically in early childhood [7,8]. Reactivation of the latent virus into lytic replication to
allow shedding and transmission of the virus
probably occurs in vivo as memory cells differentiate further, for example, into plasma cells in
response to antigen stimulation. Thus, plasma cells
are known to harbour the EBV lytic replication
that can be detected in peripheral lymphocytes
in vivo [9].
Infection with EBV causes nave B cells to transform into proliferating blasts, which can then differentiate in vivo into long-lived resting memory B
cells through the process of the germinal centre

150
reaction. Different patterns of latent EBV gene
expression are associated with various types of
infected lymphocyte. These can be observed in
cell culture either as a result of infecting primary
resting human B cells (latency III, the growth programme) or in cell lines derived from EBV associated cancers (latency I or II) [1]. The different
latency programmes were discovered in such cell
lines but have now been related to the biology of
EBV infection in vivo.
After entry of the virus into the resting B cell,
nine latent viral proteins and two small RNAs
(the EBER RNAs) constituting the latency III or
growth programme, are expressed [10]: the proteins include EBV nuclear antigens EBNA-1,
EBNA-2, EBNA-3A, 3B, 3C and EBNA-LP as well
as the latent membrane proteins LMP-1, LMP-2A
and LMP-2B (Table 1). As a result, nave B cells
become proliferating blasts. The virus protein
expression later becomes restricted to the latency
II programme [10,11] in which only EBNA-1,
LMP-1, LMP-2A and the EBER RNAs are
expressed. These two latent membrane proteins
are thought to produce the signal for the latently
infected B cell blasts to form germinal centres
[12], which causes the germinal centre B cells to
differentiate into latently infected memory B cells.
The virus enters latent persistence and shuts down
the expression of viral proteins [13], when only
LMP-2A is possibly expressed [14,15]. Latently
infected memory cells circulate between Waldeyers ring and the peripheral blood without
being detected by the immune system [16]. Even
during infectious mononucleosis, where a large
number of B cells carry EBV, no infected proliferating B blasts are found in the blood, only memory
cells [17]. This could be due to EBV actively avoiding immune detection or just efcient killing of
EBV infected B blasts by T cells in the peripheral
blood.
In latently infected memory B cells, the virus
remains silent. Only when memory B cells divide,
a natural process to maintain memory cell numbers, is EBNA-1 expressed [13], allowing the viral
genome to replicate alongside the host chromosomes.
The process of establishing a latent EBV infection by transforming nave B cells into resting
memory cells in germinal centres mimics the natural process of B-cell activation in response to a
foreign antigen [18,19]. In immune-activated B
Copyright # 2004 John Wiley & Sons, Ltd.

W. Amon and P. J. Farrell

cells, antigen-binding leads to the transition to
becoming a memory B cell but in an EBV-driven
cell, the viral proteins provide the signals necessary for this process. Lymphoblasts resulting
from antigen activation show a similar cell surface
phenotype [2022] and morphology [23] to those
produced by EBV infection in cell culture.
EBV GENE EXPRESSION IN EBV
ASSOCIATED CANCERS
EBV is associated with the development of both Bcell and epithelial cell malignancies. These include
Burkitts lymphoma [24], nasopharyngeal carcinoma [25] and Hodgkins disease [26] as well
as cancers in immunocompromised hosts like
post-transplantation lymphoproliferative disease
(PTLD) [27] and AIDS-associated lymphomas
[28], all reviewed in [1]. About 5%10% of gastric
carcinomas are also associated with EBV.
Immunoblastic lymphomas display a type III
latency pattern and in many ways resemble lymphoblastoid cell lines (LCLs) that result from infection of resting B cells in vitro. The powerful CTL
mediated immune surveillance directed against
EBV proteins expressed in latency III normally
kills such cells so they are not detected in the peripheral circulation. Immunosuppression provides
the opportunity for latency III cells to develop
into immunoblastic lymphoma [29]. Approximations to latency II (EBV protein expression limited
to EBNA-1 and LMPs) are found in nasopharyngeal carcinoma, gastric carcinoma, T cell lymphomas and Hodgkins disease (B cell lymphoma)
[3035]. In latency I, EBNA-1 is the only EBV protein expressed and this is similar to the pattern
observed in vivo as memory cells occasionally
divide. Burkitts lymphoma (BL) cells display
this phenotype [22,36,37].
BURKITTS LYMPHOMA CELL LINES
PROVIDE A MODEL FOR LATENCY IN VITRO
Cell lines derived from Burkitts lymphomas such
as the Akata cell line [38] are useful for studying
latency and reactivation in vitro. Akata cells have
a latency I infection and the EBV can be reactivated by cross-linking the surface immunoglobulin (B cell receptor, BCR) with an anti-Ig
antibody, so as to mimic the binding of antigen
to the BCR [39,40]. This is the most rapid and efcient way to induce the EBV lytic cycle in vitro and
may reect physiological mechanisms that operate
Rev. Med. Virol. 2005; 15: 149156.

Copyright # 2004 John Wiley & Sons, Ltd.

Genome maintenance
(binds origin of latent replication)
Transcription factor, binds RBP-J,
activates cellular and viral genes
Like other EBNA3s balances EBNA-2
effect on RBP-J, binds CtBP
Transcriptional regulator
Overcomes cell cycle
checkpoints, binds CtBP
Co-activates EBNA-2 responsive genes,
increases efciency of immortalisation
Activates NF-B, homologue to CD40,
prevents apoptosis
Inhibitor of BCR signalling,
blocks viral lytic cycle,
provides survival signals
Function unclear
Role in tumourigenicity of
Burkitts lymphoma cells
Function unclear, may express
RPMS1 and A73 proteins

()

Immunoblastoid
lymphomas
(Latency III)

Hodgkins
disease, NPC
(Latency II)

NPC, nasopharyngeal carcinoma; EBNA, Epstein-Barr virus nuclear antigen; LMP, latent membrane protein; EBER, Epstein-Barr virus-encoded small
RNA; BARTs, Bam A rightward transcripts.

LMP2B
EBER
RNAs
BARTs

LMP2A

LMP1

EBNA-LP

EBNA-3B
EBNA-3C

EBNA-3A

EBNA-2

EBNA-1

Gene function

Burkitts
lymphoma
(Latency I)

Required for
immortalisation

Table 1. Function and expression of Epstein-Barr virus latent genes

Reactivation of EBV from latency

151

Rev. Med. Virol. 2005; 15: 149156.

152

Figure 1. Overview of EBV lytic cycle. The lytic cycle can be

induced with anti-IgG, which cross-links the B cell receptor to
mimic antigen binding. Immediate early, early and late genes
are expressed in sequential order. Late gene expression is prevented by inhibitors of lytic DNA replication such as acyclovir

as latently infected memory cells differentiate

towards the plasma cell stage. Some BL cell lines
can alternatively be reactivated by treatment with
transforming growth factor (TGF-) [41] but the
TGF- signalling pathways are defective in many
BL lines [42]. The lack of expression of LMP proteins in latency I is important for allowing reactivation via the BCR; these proteins normally
prevent reactivation by interfering with signal
transduction from the BCR [43].
Viral gene expression in the lytic cycle follows a
temporal and sequential order (Figure 1). The
immediate early genes are followed by early and
late genes. In EBV virology, these terms are used
to describe stages of gene expression in reactivation. The commonly used antiviral agents acyclovir and ganciclovir block the viral lytic cycle
DNA replication [44,45] but only do this when
they have been activated by phosphorylation by
kinases expressed by the virus, accounting for
their specicity for virus infected cells [46]. These
agents are only effective against lytic replicating
virus and have no activity on latent infection.
Viral immediate early genes are induced
directly by signal transduction from the BCR,
independent of the expression of other proteins.
Two immediate early mRNAs have been reported
for EBV [47] encoding the proteins BZLF1 and
BRLF1 but BZLF1 appears to be the major immediate early protein in EBV. The systematic nomenclature of EBV genes is based on position and
Copyright # 2004 John Wiley & Sons, Ltd.

W. Amon and P. J. Farrell

orientation in the genome, for example, BZLF1 is
the rst leftward gene starting in the BamHI Z
region [47]. The product of the BZLF1 gene is
also known as Zta, ZEBRA and EB1. Expression
of BZLF1 alone is sufcient to trigger the entire
lytic cascade [48,49]. BZLF1 is a transcription factor with some sequence similarity to c-Fos and
has binding sites in several viral early gene promoters and promoters of cellular genes, as well as the
lytic origins of replication. BZLF1 further activates
its expression by binding to its own promoter and
activates the adjacent gene BRLF1, which is also a
transcription factor [50]. Together BZLF1 and
BRLF1 activate most of the early genes in the
next phase of the lytic cycle. Although there will
most likely be several blocks that have to be overcome to complete the lytic cycle, the induction of
the Zp promoter appears to be the primary event
in the reactivation of EBV via signal transduction
from the BCR. BZLF1 has also been shown to
halt cell cycle progression in some tumour cell
lines [51].
To study its regulation, the complete BZLF1
gene with its promoter (Zp) was cloned into a plasmid containing the EBV origin of replication and
the EBNA1 gene [52,53]. After transfection into
Akata cells, such plasmids are maintained in the
same way as the normal EBV genome plasmid.
In the cloned plasmids the regulation of the
BZLF1 gene was shown to be the same as the normal BZLF1 gene in the complete viral genome.
This provided a system in which it was possible
to undertake detailed mutagenesis of the Zp promoter to identify the sequences that mediate the
induction of Zp in response to signal transduction
from the BCR. Cell transcription factors that bind
to these DNA sequences in Zp were shown to be
modied in response to the signal transduction
and this provides the current molecular description of how reactivation of Zp and the BZLF1
gene occurs in the latent viral genome (Figure 2).
Zp is kept inactive by a repressive chromatin
structure that is relieved by histone acetylation
caused by histone acetyl transferase recruited by
MEF-2D that has become dephosphorylated as a
result of the signal transduction from the BCR
[5254]. Additional repression of the latent viral
genome occurs through DNA methylation at
CpG dinucleotides that tends to accumulate in
transcriptionally inactive chromatin but the complete regulation of Zp could be reconstituted on
Rev. Med. Virol. 2005; 15: 149156.

Reactivation of EBV from latency

Figure 2. Activation of the immediate early promoter Zp through

B-cell receptor signalling. Upon activation the B cell receptor
(BCR) is phosphorylated by protein tyrosine kinases. Engagement of three major signalling pathways leads to the activation
of several transcription factors on Zp. The key events include
dephosphorylation of MEF2D and phosphorylation of CREB/
ATF. This induces histone acetylation and relief of repressive
chromatin structures. BZLF1 binds and activates its own promoter. PLC, phospholipase C; IP3, inositol trisphosphate; CaMK, calmodulin-dependent kinase; CaN, calcineurin; JNK, c-Jun aminoterminal kinase; MEK, MAPK kinase; MAPK, mitogen-activated
protein kinase; MEF2D, myocyte enhancer factor 2D; ATF, activating transcription factor; CREB, cAMP response element binding
protein; ZI/ZII/ZIII/ZV, promoter elements of Zp

the plasmids in the absence of any detectable DNA

methylation [52].
NOVEL THERAPEUTIC APPROACHES FOR
EBV ASSOCIATED CANCERS BASED ON
REACTIVATION OF THE LATENT
VIRAL GENOME
In addition to the specic physiological pathway
of reactivation that has been discussed so far, there
are several other non-specic ways of causing
induction of the EBV lytic cycle in latently infected
cells. For example treatment of infected cell lines
with PMA (phorbol 12-myristate 13-acetate) [55]
or butyrate [56] will induce virus replication in
many cases. In some BL cell lines, treatment with
the methylation inhibitor 5-azacytidine causes
induction of the lytic cycle at rather low efciency
[57]. This is particularly interesting because 5-azacytidine is licensed for therapeutic use in cancer
treatment. It was therefore used in a preliminary
Copyright # 2004 John Wiley & Sons, Ltd.

153
trial in patients with nasopharyngeal carcinoma
to test whether it might cause reactivation of the
latent EBV genome and tumour cell death [58].
In fact there was little effect but this was the rst
clinical attempt to apply the strategy of using the
presence of EBV in the tumour cells to target therapy to this type of cancer. Further development of
the strategy will require identication of novel
compounds that can specically cause reactivation
of the EBV lytic cycle in latently infected tumour
cells and these compounds will have to be acceptable for clinical use.
The detailed understanding of the regulation of
Zp and induction of BZLF1 may provide ways to
identify novel compounds that could be used to
induce the early part of the lytic cycle. For example
an Akata cell line containing the Zp luciferase
reporter plasmids has been adapted into a 96well plate format high throughput screening assay
for such compounds [53]. In several of the EBV
associated cancers there is expression of the LMP
proteins, which can prevent the BCR signalling
to Zp [43]. In that case it would be necessary
to identify lytic cycle inducers which acted closer
to the EBV gene regulation itself, downstream
of the signal transduction pathway or even
acted less specically to reactivate the virus (like
butyrate, which non-specically causes histone
deacetylation).
Another approach to induction of the lytic cycle
in EBV-positive tumour cells is transfection with a
BZLF1 expression vector. Introduction of modied
adenovirus vectors expressing EBV early genes led
to the induction of the lytic cycle of EBV and inhibited tumour growth in Burkitts lymphomas and
nasopharyngeal carcinomas grown in nude mice
[59,60]. However, this approach is limited by the
efciency of the gene delivery methods currently
available.
To avoid the problem of inducers of the lytic
cycle causing an EBV viraemia, one could block
EBV DNA replication by giving patients acyclovir.
There has been an ingenious renement of this in
an experimental animal model test of the strategy
that considerably improved the killing of tumour
cells. Investigation of the mechanism by which
the chemotherapy agent cisplatin kills gastric carcinoma cells harbouring EBV in experimental animals revealed that the cisplatin somehow induces
the EBV lytic cycle and this probably plays a role
in the tumour cell death. By also co-administering
Rev. Med. Virol. 2005; 15: 149156.

154
ganciclovir with the cisplatin, improved tumour
cell killing was achieved [61]. The mechanism
probably involves the toxic effects of ganciclovir,
activated by the EBV lytic cycle in some of the
tumour cells, then diffusing and killing bystander
cells in the tumour. A similar result was achieved
in LCLs when combining the chemotherapeutic
drugs gemcitabine or doxorubicin with ganciclovir
[62].
GENERAL APPROACH
Reactivation of the latent viral genome in EBV
associated cancers, causing cancer cell death or
recognition by the immune system is just one
example of the general strategy of targeting therapy based on the selective presence of the virus
in tumour cells. Immunotherapy, by boosting the
immune response to antigens naturally expressed
by the virus in a virus-associated cancer, is being
tested for several viruses, including EBV and
HPV [6365] and adoptive transfer of EBV specic
T cells has also been used to treat some lymphomas successfully. Other possibilities are also being
investigated, for example, the targeted loss of the
viral episome through treatment with low-dose
hydroxyurea [66] or modied adenovirus vectors
containing the EBV oriP origin of replication that
would selectively be toxic or replicate in EBV
infected cells [67,68]. In fact any property of the
virus that is active in the tumour cells could provide the selectivity to target therapywe just
have to be sufciently inventive to think of ways
to take advantage of it!
REFERENCES
1. Rickinson AB, Kieff E. Epstein-Barr virus. In Fields
Virology, Howley PM (ed.). Lippincott-Raven:
Philadelphia, 1996; 23972446.
2. Baer R, Bankier AT, Biggin MD, et al. DNA sequence
and expression of the B958 Epstein-Barr virus genome. Nature 1984; 310: 207211.
3. de Jesus O, Smith PR, Spender LC, et al. Updated
Epstein-Barr virus (EBV) DNA sequence and analysis of a promoter for the BART (CST, BARF0) RNAs
of EBV. J Gen Virol 2003; 84: 14431450.
4. Hammerschmidt W, Mankertz J. Herpesviral DNA
replication: between the known and the unknown.
Semin Virol 1991; 64: 257269.
5. Niederman JC, Miller G, Pearson HA, Pagano JS,
Dowaliby JM. Infectious mononucleosis. EpsteinBarr-virus shedding in saliva and the oropharynx.
N Engl J Med 1976; 294: 13551359.

Copyright # 2004 John Wiley & Sons, Ltd.

W. Amon and P. J. Farrell

6. Crawford DH. Biology and disease associations of
Epstein-Barr virus. Philos Trans R Soc Lond B Biol
Sci 2001; 356: 461473.
7. Babcock GJ, Decker LL, Volk M, Thorley-Lawson
DA. EBV persistence in memory B cells in vivo.
Immunity 1998; 9: 395404.
8. Miyashita EM, Yang B, Lam KM, Crawford DH,
Thorley-Lawson DA. A novel form of Epstein-Barr
virus latency in normal B cells in vivo. Cell 1995; 80:
593601.
9. Thorley-Lawson DA, Gross A. Persistence of the
Epstein-Barr virus and the origins of associated lymphomas. N Engl J Med 2004; 350: 13281337.
10. Thorley-Lawson D. Epstein-Barr virus: exploiting
the immune system. Nature Rev Immunol 2001; 1:
7582.
11. 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 USA 2000; 97: 12250
12255.
12. Casola S, Otipoby KL, Alimzhanov M, et al. B cell
receptor signal strength determines B cell fate. Nat
Immunol 2004; 5: 317327.
13. Hochberg D, Middeldorp JM, Catalina M, Sullivan
JL, Luzuriaga K, Thorley-Lawson DA. Demonstration of the Burkitts lymphoma Epstein-Barr virus
phenotype in dividing latently infected memory cells
in vivo. Proc Natl Acad Sci USA 2004; 101: 239244.
14. Qu L, Rowe DT. Epstein-Barr virus latent gene
expression in uncultured peripheral blood lymphocytes. J Virol 1992; 66: 37153724.
15. Miyashita EM, Yang B, Babcock GJ, Thorley-Lawson
DA. Identication of the site of Epstein-Barr virus
persistence in vivo as a resting B cell. J Virol 1997;
71: 48824891.
16. Laichalk LL, Hochberg D, Babcock GJ, Freeman RB,
Thorley-Lawson DA. The dispersal of mucosal memory B cells: evidence from persistent EBV infection.
Immunity 2002; 16: 745754.
17. Hochberg D, Souza T, Catalina M, Sullivan JL,
Luzuriaga K, Thorley-Lawson DA. Acute infection
with Epstein-Barr virus targets and overwhelms
the peripheral memory B-cell compartment with
resting, latently infected cells. J Virol 2004; 78:
51945204.
18. MacLennan IC, Liu YL, Ling NR. B cell proliferation
in follicles, germinal centre formation and the site of
neoplastic transformation in Burkitts lymphoma.
Curr Top Microbiol Immunol 1988; 141: 138148.
19. Liu YJ, Arpin C. Germinal center development.
Immunol Rev 1997; 156: 111126.
20. Thorley-Lawson DA, Schooley RT, Bhan AK, Nadler
LM. Epstein-Barr virus superinduces a new human B

Rev. Med. Virol. 2005; 15: 149156.

Reactivation of EBV from latency

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

cell differentiation antigen (B-LAST 1) expressed on

transformed lymphoblasts. Cell 1982; 30: 415425.
Thorley-Lawson DA, Nadler LM, Bhan AK,
Schooley RT. BLAST-2 [EBVCS], an early cell surface
marker of human B cell activation, is superinduced
by Epstein Barr virus. J Immunol 1985; 134: 3007
3012.
Rowe M, Rowe DT, Gregory CD, et al. Differences in
B cell growth phenotype reect novel patterns of
Epstein-Barr virus latent gene expression in Burkitts
lymphoma cells. EMBO J 1987; 6: 27432751.
Nilsson K. The nature of lymphoid cell lines and
their relationship to the virus. In The Epstein-Barr
Virus, Achong BG (ed.). Springer-Verlag: Berlin,
1979; 225281.
Epstein MA, Achong BG, Barr YM. Virus particles in
cultured lymphoblasts from Burkitts lymphoma.
Lancet 1964; 1: 702703.
Andersson-Anvret M, Forsby N, Klein G, Henle W,
Biorklund A. Relationship between the Epstein-Barr
virus genome and nasopharyngeal carcinoma in
Caucasian patients. Int J Cancer 1979; 23: 762767.
Weiss LM, Movahed LA, Warnke RA, Sklar J. Detection of Epstein-Barr viral genomes in Reed-Sternberg
cells of Hodgkins disease. N Engl J Med 1989; 320:
502506.
Thomas JA, Hotchin NA, Allday MJ, et al. Immunohistology of Epstein-Barr virus-associated antigens
in B cell disorders from immunocompromised individuals. Transplantation 1990; 49: 944953.
Hamilton-Dutoit SJ, Pallesen G, Franzmann MB, et al.
AIDS-related lymphoma. Histopathology, immunophenotype, and association with Epstein-Barr virus
as demonstrated by in situ nucleic acid hybridization. Am J Pathol 1991; 138: 149163.
Timms JM, Bell A, Flavell JR, et al. Target cells of
Epstein-Barr-virus (EBV)-positive post-transplant
lymphoproliferative disease: similarities to EBVpositive Hodgkins lymphoma. Lancet 2003; 361:
217223.
Niedobitek G, Kremmer E, Herbst H, et al. Immunohistochemical detection of the Epstein-Barr virusencoded latent membrane protein 2A in Hodgkins
disease and infectious mononucleosis. Blood 1997;
90: 16641672.
Herbst H, Dallenbach F, Hummel M, et al. EpsteinBarr virus latent membrane protein expression in
Hodgkin and Reed-Sternberg cells. Proc Natl Acad
Sci USA 1991; 88: 47664770.
Deacon EM, Pallesen G, Niedobitek G, et al. EpsteinBarr virus and Hodgkins disease: transcriptional
analysis of virus latency in the malignant cells.
J Exp Med 1993; 177: 339349.
Oudejans JJ, Dukers DF, Jiwa NM, et al. Expression of
Epstein-Barr virus encoded nuclear antigen 1 in

Copyright # 2004 John Wiley & Sons, Ltd.

155

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

benign and malignant tissues harbouring EBV. J Clin

Pathol 1996; 49: 897902.
Imai S, Koizumi S, Sugiura M, et al. Gastric carcinoma: monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein. Proc
Natl Acad Sci USA 1994; 91: 91319135.
Brooks L, Yao QY, Rickinson AB, Young LS. EpsteinBarr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1,
LMP1, and LMP2 transcripts. J Virol 1992; 66: 2689
2697.
Hatzubai A, Ana M, Masucci MG, et al. Downregulation of the EBV-encoded membrane protein
(LMP) in Burkitt lymphomas. Int J Cancer 1987; 40:
358364.
Gregory CD, Rowe M, Rickinson AB. Different
Epstein-Barr virus-B cell interactions in phenotypically distinct clones of a Burkitts lymphoma cell line.
J Gen Virol 1990; 71: 14811495.
Takada K, Ono Y. Synchronous and sequential activation of latently infected Epstein-Barr virus genomes. J Virol 1989; 63: 445449.
Shimizu N, Takada K. Analysis of the BZLF1 promoter of Epstein-Barr virus: identication of an antiimmunoglobulin response sequence. J Virol 1993;
67: 32403245.
Takada K. Cross-linking of cell surface immunoglobulins induces Epstein-Barr virus in Burkitt lymphoma lines. Int J Cancer 1984; 33: 2732.
Fahmi H, Cochet C, Hmama Z, Opolon P, Joab I.
Transforming growth factor beta 1 stimulates expression of the Epstein-Barr virus BZLF1 immediateearly gene product ZEBRA by an indirect mechanism which requires the MAPK kinase pathway.
J Virol 2000; 74: 58105818.
Inman G, Allday M. Apoptosis induced by TGF-b1 in
Burkitts lymphoma cells is caspase 8 dependent but
death receptor independent. J Immunol 2000; 165:
25002510.
Miller CL, Lee JH, Kieff E, Longnecker R. An integral
membrane protein (LMP2) blocks reactivation of
Epstein-Barr virus from latency following surface
immunoglobulin crosslinking. Proc Natl Acad Sci
USA 1994; 91: 772776.
Datta AK, Colby BM, Shaw JE, Pagano JS. Acyclovir
inhibition of Epstein-Barr virus replication. Proc Natl
Acad Sci USA 1980; 77: 51635166.
Packham G, Brimmell M, Cook D, Sinclair AJ, Farrell
PJ. Strain variation in Epstein-Barr virus immediate
early genes. Virology 1993; 192: 541550.
Moore SM, Cannon JS, Tanhehco YC, Hamzeh FM,
Ambinder RF. Induction of Epstein-Barr virus
kinases to sensitize tumor cells to nucleoside analogues. Antimicrob Agents Chemother 2001; 45: 2082
2091.

Rev. Med. Virol. 2005; 15: 149156.

156
47. Biggin M, Bodescot M, Perricaudet M, Farrell P.
Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells. J Virol 1987; 61: 31203132.
48. Countryman J, Miller G. Activation of expression of
latent Epstein-Barr herpesvirus after gene transfer
with a small cloned subfragment of heterogeneous
viral DNA. Proc Natl Acad Sci USA 1985; 82: 4085
4089.
49. Rooney CM, Rowe DT, Ragot T, Farrell PJ. The
spliced BZLF1 gene of Epstein-Barr virus (EBV)
transactivates an early EBV promoter and induces
the virus productive cycle. J Virol 1989; 63: 3109
3116.
50. Speck SH, Chatila T, Flemington E. Reactivation of
Epstein-Barr virus: regulation and function of the
BZLF1 gene. Trends Microbiol 1997; 5: 399405.
51. Cayrol C, Flemington EK. The Epstein-Barr virus
bZIP transcription factor Zta causes G0/G1 cell cycle
arrest through induction of cyclin-dependent kinase
inhibitors. EMBO J 1996; 15: 27482759.
52. Jenkins P, Binne U, Farrell P. Histone acetylation and
reactivation of Epstein-Barr virus from latency. J
Virol 2000; 74: 710720.
53. Binne UK, Amon W, Farrell PJ. Promoter sequences
required for reactivation of Epstein-Barr virus from
latency. J Virol 2002; 76: 1028210289.
54. Bryant H, Farrell PJ. Signal transduction and transcription factor modication during reactivation of
Epstein-Barr virus from latency. J Virol 2002; 76:
1029010298.
55. zur-Hausen H, Schulte-Holthausen H, Klein G, et al.
EBV DNA in biopsies of Burkitt tumours and anaplastic carcinomas of the nasopharynx. Nature 1970;
228: 10561058.
56. Luka J, Kallin B, Klein G. Induction of the EpsteinBarr virus (EBV) cycle in latently infected cells by
n-butyrate. Virology 1979; 94: 228231.
57. Ben-Sasson SA, Klein G. Activation of the EpsteinBarr virus genome by 5-aza-cytidine in latently
infected human lymphoid lines. Int J Cancer 1981;
28: 131135.
58. Chan AT, Tao Q, Robertson KD, et al. Azacitidine
induces demethylation of the Epstein-Barr virus genome in tumors. J Clin Oncol 2004; 22: 13731381.

Copyright # 2004 John Wiley & Sons, Ltd.

W. Amon and P. J. Farrell

59. Westphal EM, Mauser A, Swenson J, Davis MG,
Talarico CL, Kenney SC. Induction of lytic EpsteinBarr virus (EBV) infection in EBV-associated malignancies using adenovirus vectors in vitro and in vivo.
Cancer Res 1999; 59: 14851491.
60. Feng WH, Westphal E, Mauser A, et al. Use of adenovirus vectors expressing Epstein-Barr virus (EBV)
immediate-early protein BZLF1 or BRLF1 to treat
EBV-positive tumors. J Virol 2002; 76: 1095110959.
61. Feng WH, Israel B, Raab-Traub N, Busson P, Kenney
SC. Chemotherapy induces lytic EBV replication and
confers ganciclovir susceptibility to EBV-positive
epithelial cell tumors. Cancer Res 2002; 62: 19201926.
62. Feng WH, Hong G, Delecluse HJ, Kenney SC. Lytic
induction therapy for Epstein-Barr virus-positive Bcell lymphomas. J Virol 2004; 78: 18931902.
63. Santin AD, Hermonat PL, Ravaggi A, et al. Interleukin-10 increases Th1 cytokine production and cytotoxic potential in human papillomavirus-specic
CD8( ) cytotoxic T lymphocytes. J Virol 2000; 74:
47294737.
64. Rooney CM, Roskrow MA, Smith CA, Brenner MK,
Heslop HE. Immunotherapy for Epstein-Barr virusassociated cancers. J Natl Cancer Inst Monogr 1998;
23: 8993.
65. Chapman AL, Rickinson AB, Thomas WA, Jarrett
RF, Crocker J, Lee SP. Epstein-Barr virus-specic
cytotoxic T lymphocyte responses in the blood and
tumor site of Hodgkins disease patients: implications for a T-cell-based therapy. Cancer Res 2001; 61:
62196226.
66. Chodosh J, Holder VP, Gan YJ, Belgaumi A, Sample
J, Sixbey JW. Eradication of latent Epstein-Barr virus
by hydroxyurea alters the growth-transformed cell
phenotype. J Infect Dis 1998; 177: 11941201.
67. Kenney S, Ge JQ, Westphal EM, Olsen J. Gene therapy strategies for treating Epstein-Barr virus-associated lymphomas: comparison of two different
Epstein-Barr virus-based vectors (see comments).
Hum Gene Ther 1998; 9: 11311141.
68. Hirai H, Satoh E, Osawa M, et al. Use of EBV-based
vector/HVJ-liposome complex vector for targeted
gene therapy of EBV-associated neoplasms. Biochem
Biophys Res Commun 1997; 241: 112118.

Rev. Med. Virol. 2005; 15: 149156.