Microbes and Infection 4 (2002) 853–857

www.elsevier.com/locate/micinf

Review

Molecular mechanisms of B-lymphocyte transformation

by Epstein–Barr virus
Gail A. Bishop a,b,c,*, Lisa K. Busch b
a
Departments of Microbiology and Internal Medicine, 3-570 Bowen Science Bldg., The University of Iowa, Iowa City, IA 52242, USA
b
Graduate Program in Molecular Biology, The University of Iowa, Iowa City, IA 52242, USA
c
VA Medical Center, Iowa City, IA 52242, USA

Abstract

The B-lymphotropic virus Epstein–Barr virus (EBV) has been implicated in the pathogenesis of B-cell malignancies, particularly in
immunodeficient individuals. This review provides a brief overview of the EBV-encoded proteins involved in B-cell transformation, and
the current state of knowledge about their roles in this process. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights
reserved.

Keywords: B lymphocyte; Epstein–Barr virus; Cell transformation; Signal transduction; Lymphoma

1. Introduction has led to considerable effort to understand the molecular

Epstein–Barr virus (EBV) is a human herpesvirus with
tropism for B lymphocytes, although it can also infect
epithelial cells. In a healthy individual, EBV establishes and
maintains latency in a relatively small number of memory B
cells, remaining quiescent. However, viral inactivity is
strongly dependent upon the continuous activity of cytolytic
CD8+ T lymphocytes (CTLs), and infected hosts also have
serum antibodies to proteins expressed in the viral lytic
cycle (reviewed in [1]). Thus, conditions resulting in immu-
nosuppression allow EBV to promote unregulated
B-lymphocyte proliferation and/or survival. B-cell tumors
directly associated with EBV-mediated transformation arise
in pharmacologically immunosuppressed patients (such as
transplant recipients), HIV-infected patients, and persons
with the genetic disorder X-linked lymphoproliferative
disease (XLP). In addition, EBV has been implicated as
contributing to the pathogenesis of Burkitt’s lymphoma and
Hodgkin’s disease, although the specific role played by the
virus in these tumors is not yet known (reviewed in [1]). The
strong potential of EBV to contribute to neoplastic disease
* Corresponding author. Tel.: +1-319-335-7945; fax: +1-319-335-9006.
E-mail address: gail-bishop@uiowa.edu (G.A. Bishop).
© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
PII: S 1 2 8 6 - 4 5 7 9 ( 0 2 ) 0 1 6 0 5 - 2
mechanisms by which EBV transforms B cells. This review
will focus upon a concise summary of the state of knowl-
edge about these mechanisms.
2. EBV-associated B-cell malignancies
While the majority of EBV-infected individuals will
experience no symptoms from latent viral infection, EBV
has been associated with several human malignancies, most
notably, endemic Burkitt’s lymphoma in sub-Saharan Af-
rica, nasopharyngeal carcinoma in southeast Asia, and
immunoblastic B-cell lymphoma in immunocompromised
individuals; in these tumors, EBV is present in almost all
cases (reviewed in [2]). A causal relationship between EBV
and the tumors is strongly suggested by the prevalence of
the association and the clonal nature of both EBV and the
tumor cells. EBV is also frequently involved in the patho-
genesis of the genetic disorder XLP. This disease was first
identified in the 1970s in a family where several young male
children died from a fatal lymphoproliferative disease
resembling a dysregulated form of infectious mononucleo-
sis, an acute and self-limiting illness caused by primary
EBV infection in healthy individuals (reviewed in [3]). The
gene encoding the mutant protein involved in XLP, signal-
ing lymphocyte activation molecule-association protein
(SAP), has recently been cloned and is a small signaling
854 G.A. Bishop, L.K. Busch / Microbes and Infection 4 (2002) 853–857
protein containing a single Src homology 2 (SH2) domain,
believed to function as a negative regulator in both B- and
T-cell signaling (reviewed in [3]). SAP may play a general
role in immune regulation as it has recently been reported
that XLP individuals can exhibit symptoms prior to EBV
infection (ibid.).
3. EBV immortalizing proteins
3.1. Overview
During in vitro infection, EBV transforms primary B
lymphocytes into semi-activated lymphoblastoid cell lines
(LCLs). EBV encodes >85 genes, but only nine viral pro-
teins are expressed by LCLs (reviewed in [2]). They include
the Epstein–Barr nuclear antigens (EBNA) 2, 3A, 3B, 3C
and LP, which are important transcriptional regulators for
both viral and cellular proteins, EBNA1, which is respon-
sible for the maintenance of the viral episome during
cellular division, latent membrane protein (LMP) 1, which
mimics an activated receptor of the tumor necrosis factor
receptor (TNF-R) super-family; and LMP2A, which con-
tains immunoreceptor tyrosine-based activation motifs
(ITAMs) and blocks certain signals transduced via the
B-cell antigen receptor (BCR). LMP2B, a variant of
LMP2A produced from a 3' promoter that lacks the amino-
terminal ITAMs, is also expressed and is believed to
down-modulate LMP2A effects. EBNA2, 3A, 3B, and 3C
exhibit allelic polymorphisms, allowing EBV isolates to be
classified as type 1 or 2 (reviewed in [4]). Immortalized B
cells also transcribe two non-translated, non-polyadenylated
RNAs, EBV early RNA (EBER) 1 and 2, which lack known
functions but are commonly used as markers of EBV
infection because of their abundance. Of these nine proteins,
EBNA1, 2, 3A, 3C and LMP1 are required for EBV-
mediated B-cell transformation [5].
3.2. EBNA1
EBNA1 is the only EBV protein detected in all EBV-
associated tumors and is required for the maintenance of the
EBV episome [6]. The amino terminal half of EBNA1
contains a 239-amino acid region of repeating glycine–gly-
cine–alanine residues which inhibits proteosomal degrada-
tion and presentation to CTLs [7], and two linking regions
surrounding the gly–ala repeat region that mediate homo-
typic interactions between DNA bound EBNA1 molecules
[8]. The linking regions of EBNA1 are required for episo-
mal replication, maintenance, and transcription, supporting
the hypothesis that episomally bound EBNA1 links to
EBNA1 bound to mitotic chromosomes, resulting in the
constant number of episomes found in infected cell lines
over time [8]. The importance of EBNA1 as a transcrip-
tional enhancer is unknown, but it may contribute to cell
transformation, as mice carrying an EBNA1 transgene
driven by the immunoglobulin heavy chain
promoter/enhancer die from B-cell lymphomas [9].
3.3. EBNA2
EBNA2 is a potent transcriptional activator of viral and
cellular genes and is one of the first EBV proteins detected
in infection; it is required for B-cell immortalization in vitro
and is the main determinant in transformation efficiency
between the EBV serological types 1 and 2 (reviewed in
[4]). EBNA2 from the prototype 1 EBV strain B95-8
contains a polyproline region, a glycine–arginine repeat
region, and a carboxyl terminal acidic transactivation do-
main that interacts with basal transcription factors [10].
Unlike EBNA1, EBNA2 does not bind DNA directly, but is
recruited to DNA by the cellular proteins Cp binding
factor/recombination site binding protein-Jj, Spi-1/PU.1,
and perhaps other as yet uncharacterized factors [11].
Similar to EBNA1, EBNA2 in the absence of other EBV
proteins can be oncogenic, as 90% of transgenic mice
expressing EBNA2 from the SV40 early enhancer-promoter
develop kidney tumors [12].
3.4. EBNA3s and EBNA-LP
EBNA3A, 3B and 3C are encoded by three adjacent
genes in the EBV genome that originally arose by gene
duplication [4]. The proteins retain homology only in the
amino-terminal region which is capable of interacting with
Cp binding factor, just as EBNA2 does. Genetic analysis
reveals that EBNA3B is not required for immortalization,
while EBNA3A and 3C are essential [13]. All three proteins
are able to inhibit EBNA2 transactivation in transient
transfection assays, but their exact role in vivo remains
unclear, as the inhibitory effect of EBNA3C depends upon
the promoter being examined [4]. EBNA-LP is not required
for EBV immortalization of B cells, but its presence
enhances transformation [5]. EBNA-LP can enhance
EBNA2-mediated transcriptional activation by 10- to 100-
fold. There has been one report of EBNA-LP reorganizing
the position of EBNA3A in the nucleus, suggesting the
possibility of a complex transcriptional regulatory network
of EBNAs [4].
3.5. LMP1
The role of LMP1 in EBV-mediated oncogenesis has
been intensively studied, both in epithelial cells and in B
lymphocytes, the latter being the cell type of focus in this
review. LMP1 is expressed in the majority of EBV-
associated malignancies, and is critical to the transformation
of B cells in vitro [14]. Additionally, transgenic expression
of LMP1 driven by an immunoglobulin promoter/enhancer
in mice accelerates development of age-related B-cell tu-
mors, in the absence of additional EBV-encoded proteins
[15]. Thus, while LMP1 expression alone cannot transform
 G.A. Bishop, L.K. Busch / Microbes and Infection 4 (2002) 853–857
primary human B cells [16], it is clearly essential to the
process, in which it plays a major role. LMP1 consists of
short N-terminal and long C-terminal CY domains, sepa
rated by six membrane-spanning domains; the latter can
self-aggregate and initiate LMP1 signaling. However, the
C-terminal CY domain is both necessary and sufficient to
mediate LMP1 signaling to B cells [17]. LMP1 expression
in B cells markedly mimics the effects of ligation of an
important B-cell activation receptor, CD40, and both CD40
and LMP1 associate with members of the TNF-R-associated
factor (TRAF) family of cytoplasmic adapter proteins (re-
viewed in [18]). The ability of LMP1 to signal constitutively
via self-aggregation is likely to contribute to its oncogenic
potential, an idea supported by a recent study of the
interaction between LMP1 and lipid rafts [19], but recent
studies from several laboratories suggest that differences
between CD40 and LMP1-mediated signaling extend be-
yond the manner in which the signal is initiated. Both CD40
and LMP1 induce rapid activation of stress-activated pro-
tein kinases c-jun kinase and p38, activation of the NF-jB
family of transcription factors, and downstream B-cell
activation events that include upregulation of cell surface
antigens, protection from apoptosis, and induction of IL-6
production (reviewed in [18]). In addition, both CD40 and
LMP1 recruit TRAFs 1, 2 and 3 to lipid rafts in B cells
[20,21]. However, it was recently shown that shortly fol-
lowing this recruitment to CD40, TRAFs 2 and 3 undergo
degradation, while LMP1-mediated recruitment is not asso-
ciated with TRAF degradation [21]. This difference in
TRAF regulation may contribute to LMP1’s transforming
properties, as analysis of hybrid receptors revealed that
signaling via the LMP1 CY domain provides amplified and
sustained B-cell activation, as measured by the parameters
listed above [21]. Interestingly (and of potential therapeutic
relevance), CD40 signals mediate TRAF degradation even
in cells expressing LMP1 [21], demonstrating that LMP1
does not block TRAF degradation, but cannot initiate this
process. A contributing factor to this difference may be the
binding affinity of individual TRAFs to each receptor.
Recent experiments studying endogenous TRAFs in B
lymphocytes [22] have confirmed results of earlier studies
using exogenously overexpressed proteins in epithelial
cells, or bacterially expressed fusion proteins [23], showing
that while CD40 binds TRAF2 strongly, TRAF2 association
with LMP1 is much weaker. Additionally, it is becoming
increasingly clear that while B-cell transformation and
signaling by LMP1 is at least partially TRAF-dependent, it
also has important TRAF-independent components [22,24].
One of these components has been reported to be the
adapter protein TNF-R-associated death domain-containing
protein (TRADD); however, this association cannot be
confirmed to occur between LMP1 and normal levels of
TRADD in B lymphocytes [22], and it is likely that
additional LMP1-binding cellular proteins remain to be
identified.
855
3.6. LMP2A
The effects of LMP2 expression on B-cell physiology are
multiple and complex. Like LMP1, LMP2A appears to
exploit the signaling pathways of a normal B-cell receptor,
in this instance the B-cell antigen receptor (BCR). It has
been shown that EBV recombinant viruses can transform
cultured cells in the absence of functional LMP2 [25],
unlike the proteins discussed above. However, LMP2 inter-
acts with a critical normal B-cell signaling pathway, and, as
detailed below, LMP2A is likely to participate at least
indirectly in the process of B-cell lymphomagenesis asso-
ciated with EBV reactivation. Like the CD79a and CD79b
molecules of the BCR complex, LMP2A contains two
ITAMs, and can bind the Src family kinase Lyn, via these
motifs [25]. Expression of LMP2A has been shown to block
both B-cell proliferation and Ca2+ mobilization stimulated
by BCR crosslinking [1,25]. Association of LMP2A with
Lyn has also been demonstrated to inhibit BCR transloca-
tion to lipid rafts [26], and to promote ubiquitination of
BCR-associated tyrosine kinases [27]. These effects have
recently been attributed to regulation of an SLP-65/CrkL
signaling pathway [28]. Such information suggests that
LMP2A’s principal role might be in the maintenance of
EBV latency, by preventing EBV reactivation and lytic
infection. However, in addition to blocking BCR signaling,
LMP2A can substitute for a BCR-mediated survival signal
in B cells lacking membrane immunoglobulin [29]. Inas-
much as evidence has been presented that the primary
reservoir for latent infection of EBV is memory B cells
(reviewed in [1]), LMP2A signaling to such cells could
promote their survival in the germinal center, even in the
absence of selection based upon BCR affinity. Thus,
LMP2A may serve not to directly mediate B-cell transfor-
mation, but to provide a reservoir of transformable, latently
infected cells.
4. Conclusions
Transformation of B lymphocytes by EBV is clearly a
complex and multi-step process, to which a number of
EBV-encoded proteins contribute in a variety of ways. Table
1 summarizes the known activities of each of the EBV-
transforming proteins. In addition to these activities, the
final outcome of transformation is undoubtedly influenced
by the activation status of the host B lymphocyte, and by
signals received from endogenous B-cell receptors. Main-
tenance of, and reactivation from, EBV latency is consid-
erably influenced by these parameters (for recent reviews,
see [1,30]). EBNA1, by inhibiting degradation that allows
viral peptides access to MHC class I molecules in the
endoplasmic reticulum, inhibits effective antigen presenta-
tion needed for an effective CTL response to EBV, and also
appears to have uncharacterized tumorigenic effects, at least
when expressed as a transgene. The roles of the EBNA3s
856 G.A. Bishop, L.K. Busch / Microbes and Infection 4 (2002) 853–857
Table 1
EBV proteins contributing to B-cell transformation
Protein Function
EBNA1 Episome replication and maintenance
EBNA2 Transcriptional enhancer
EBNA3, 3A Transcriptional repressor
EBNA3B, 4 Transcriptional repressor
EBNA3C, 6 Transcriptional repressor
EBNA-LP, 5 Cooperates with EBNA2
LMP1 Mimics an amplified and sustained CD40 signal
LMP2A Blocks and/or substitutes for BCR signals
LMP2B Modulates LMP2A function
.
and LP are unclear, but these proteins appear to enhance
EBNA2 functions, and contribute to transformation. As
described above, EBV proteins such as LMP2A and LMP1
mimic the normal B-cell receptors BCR and CD40, respec-
tively. In the case of LMP2A, which also inhibits signaling
through the endogenous BCR, activity of the viral protein
could allow B cells to escape normal BCR-mediated cell
cycle arrest and apoptosis. While LMP1 does not block
CD40 signaling, its constitutive activity and inability to
regulate TRAF function normally could contribute to con-
tinuous and abnormal survival of B cells. Finally, the BCR
costimulatory receptor CD21 serves as a cell surface recep-
tor for EBV, so EBV binding to this receptor may also affect
B-cell activation status and the regulation of B-cell survival.
It is clear that a more detailed understanding of how each of
the EBV transforming proteins functions in B lymphocytes
is desirable. The demonstrated ability of CD40 to mediate
TRAF degradation even in cells signaling through LMP1
also suggests that such knowledge may ultimately be useful
for intervention in EBV-mediated B-cell transformation.
References
[1] D.A. Thorley-Lawson, EBV: exploiting the immune system, Nat.
Rev. Immunol. 1 (2001) 75–82.
[2] B. Wensing, P.J. Farrell, Regulation of cell growth and death by
EBV, Microbes Infect. 2 (2000) 77–84.
[3] D. Howie, J. Sayos, C. Terhorst, M. Morra, The gene defective in
XLP controls T cell dependent immune surveillance against EBV,
Curr. Opin. Immunol. 12 (2000) 474–478.
[4] D.T. Rowe, EBV immortalization and latency, Frontiers Biosci. 4
(1999) d346–371.
[5] W. Hammerschmidt, B. Sugden, Genetic analysis of immortalizing
functions of EBV in human B lymphocytes, Nature 340 (1989)
393–397.
[6] J. Yates, N. Warren, B. Sudgen, Stable replication of plasmids
derived from EBV in various mammalian cells, Nature 313 (1985)
812–815.
[7] J. Livitskaya, A. Shapiro, A. Leonchiks, A. Ciechanover, M.G. Ma-
succi, Inhibition of ubiquitin/proteasome-dependent protein degra-
dation by the gly–ala repeat domain of EBNA-1, Proc. Natl. Acad.
Sci. USA 94 (1997) 12616–12621.
[8] D. Mackey, B. Sudgen, The linking regions of EBNA1 are essential
for its support of replication and transcription, Mol. Cell. Biol. 19
(1999) 3349–3359.
[9] J.B. Wilson, J.L. Bell, A.J. Levine, Expression of EBNA-1 induces
B cell neoplasia in transgenic mice, EMBO J. 15 (1996) 3117–3126.
Required for B-cell transformation
Yes
Yes
Yes
No
Yes
No
Yes
No
No
[10] X. Tong, F. Wang, C. Thut, E. Kieff, The EBNA-2 acidic domain can
interact with TFIIB, TAF40, and RPA70 but not with TATA-binding
protein, J. Virol. 69 (1995) 585–588.
[11] G. Laux, B. Adam, L.J. Strobl, F. Moreau-Gachelin, The Spi-1/PU.1
and Spi-B ets family transcription factors and the recombination
signal binding protein RBP-Jj interact with an EBNA2-responsive
cis-element, EMBO J. 13 (1994) 5624–5632.
[12] J. Tornell, S. Farzad, A. Espander-Jansson, G. Matejka, O. Isaksson,
L. Rymo, Expression of EBNA-2 in kidney tubule cells induces
tumors in transgenic mice, Oncogene 12 (1996) 1521–1528.
[13] B. Tomkinson, K. Robertson, E. Kieff, EBNA 3A and 3C are
essential for B lymphocyte growth transformation, J. Virol. 67
(1993) 5068–5074.
[14] K.M. Kaye, K.M. Izumi, E. Kieff, EBV LMP1 is essential for
B-lymphocyte growth transformation: EBV strategy in normal and
neoplastic B cells, Proc. Natl. Acad. Sci. USA 90 (1993) 9150–9154.
[15] W. Kulwichit, R.H. Edwards, E.M. Davenport, J.F. Baskar, V. God-
frey, N. Raab-Traub, Expression of the EBV LMP1 induces B cell
lymphoma in transgenic mice, Proc. Natl. Acad. Sci. USA 95 (1998)
11963–11968.
[16] U. Zimber-Strobl, B. Kempkes, G. Marschall, R. Zeidler, C. Van
Kooten, J. Banchereau, G.W. Bornkamm, W. Hammerschmidt, EBV
LMP1 is not sufficient to maintain proliferation of B cells but both
it and activated CD40 can prolong their survival, EMBO J. 15
(1996) 7070–7078.
[17] L.K. Busch, G.A. Bishop, The EBV transforming protein, LMP1,
mimics and cooperates with CD40 signaling in B lymphocytes,
J. Immunol. 162 (1999) 2555–2561.
[18] G.A. Bishop, B.S. Hostager, Signaling by CD40 and its mimics in B
cell activation, Immunol. Res. 24 (2001) 97–109.
[19] A. Kaykas, K. Worringer, B. Sugden, CD40 and LMP-1 both signal
from lipid rafts but LMP-1 assembles a distinct, more efficient
signaling complex, EMBO J. 20 (2001) 2641–2654.
[20] B.S. Hostager, I.M. Catlett, G.A. Bishop, Recruitment of CD40,
TRAF2 and TRAF3 to membrane microdomains during CD40
signaling, J. Biol. Chem. 275 (2000) 15392–15398.
[21] K.D. Brown, B.S. Hostager, G.A. Bishop, Differential signaling and
TRAF degradation by CD40 and the EBV oncoprotein LMP1,
J. Exp. Med. 193 (2001) 943–954.
[22] L.K. Busch, G.A. Bishop, Multiple carboxyl-terminal regions of the
EBV oncoprotein, LMP1, cooperatively regulate signaling to B
lymphocytes via TRAF-dependent and TRAF-independent mecha-
nisms, J. Immunol. 167 (2001) 5805–5813.
[23] M. Sandberg, W. Hammerschmidt, B. Sugden, Characterization of
LMP1 association with TRAF1, 2 and 3, J. Virol. 71 (1997)
4649–4656.
[24] K.M. Izumi, E.C. McFarland, E.A. Riley, D. Rizzo, Y. Chen,
E. Kieff, The residues between the two transformation effector sites
of EBV LMP1 are not critical for B lymphocyte growth transfor-
mation, J. Virol. 73 (1999) 9908–9916.
 G.A. Bishop, L.K. Busch / Microbes and Infection 4 (2002) 853–857
[25] C.L. Miller, J.H. Lee, E. Kieff, R. Longnecker, An integral mem-
brane protein (LMP2) blocks reactivation of EBV from latency
following surface Ig crosslinking, Proc. Natl. Acad. Sci. USA 91
(1994) 772–776.
[26] M.L. Dykstra, R. Longnecker, S.K. Pierce, EBV coopts lipid rafts to
block the signaling and antigen transport functions of the BCR,
Immunity 14 (2001) 57–67.
[27] M. Ikeda, A. Ikeda, R. Longnecker, PY motifs of EBV LMP2A
regulate protein stability and phosphorylation of LMP2A-associated
proteins, J. Virol. 75 (2001) 5711–5718.
857
[28] N. Engels, M. Merchant, R. Pappu, A.C. Chan, R. Longnecker,
J. Wienands, EBV LMP2A employs the SLP-65 signaling module,
J. Exp. Med. 194 (2001) 255–264.
[29] R.G. Caldwell, J.B. Wilson, S.J. Anderson, R. Longnecker, EBV
LMP2A drives B cell development and survival in the absence of
normal BCR signals, Immunity 9 (1998) 405–411.
[30] K.M. Haan, A. Aiyar, R. Longnecker, Establishment of latent EBV
infection and stable episomal maintenance in murine B-cell lines,
J. Virol. 75 (2001) 3016–3020.