JOURNAL OF VIROLOGY, Aug. 2007, p. 7825–7832
0022-538X/07/$08.00⫹0 doi:10.1128/JVI.00445-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 15
MINIREVIEW
Epstein-Barr Virus Entry䌤
Lindsey M. Hutt-Fletcher*
Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center,
Louisiana State University Health Sciences Center, Shreveport, Louisiana
ATTACHMENT
(i) B cells. Eight virus glycoproteins have been implicated in
some way in EBV entry into either a B cell or an epithelial cell
(Table 1). One of the most abundant of these in the virus
envelope, gp350/220 (22), is responsible for attachment of the
virus with high affinity (36) to the complement receptor type 2
(CR2) on B cells (7, 8, 40, 41, 60, 61). An EBV recombinant
lacking gp350/220 can transform B cells with much-reduced
efficiency (20), so CR2 is perhaps not the only portal by which
the virus can access a B cell. It is, however, clearly the predominant one (Fig. 1). Antibodies to gp350/220 that block
virus binding neutralize B-cell infection, and soluble forms of
either CR2 or gp350/220 can do the same (35, 61).
Glycoprotein gp350/220 is a highly glycosylated single-pass
membrane protein, which as a result of alternative splicing is
made in two forms, with approximate masses of 350 and 220
kDa (1, 17). Residues 500 to 757, which include three repeats
of a 21-amino-acid motif with amphipathic characteristics, are
lost from the full-length 907-amino-acid protein as a result of
the splice. It does, however, maintain the reading frame, and
both forms of the protein retain the CR2 binding domain at the
amino terminus (61). Solution of the structure of an unligan-
* Mailing address: Department of Microbiology and Immunology,
Louisiana State University Health Sciences Center, 1501 Kings Highway,
Shreveport, LA 71130. Phone: (318) 675-4948. Fax: (318) 675-5764.
E-mail: [email protected].
䌤
Published ahead of print on 25 April 2007.
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ded form of gp350/220 has mapped this domain to a glycanfree surface (59) that includes a sequence with some similarity
to the natural ligand of CR2, the C3dg fragment of complement (26, 39, 61). The extracellular domain of CR2 is composed of tandem repeats of structural modules 60 to 75 amino
acids in length known as short consensus repeats (SCR), and
the binding site for gp350/220 has been genetically mapped to
a region composed of residues from the membrane distal
SCR1 and SCR2 (30). Attachment initially positions the virus
at approximately 50 nm from the cell surface (38), but the
segmental flexibility that the repeated SCRs may give to CR2
(68) and the possibility of an exchange in binding from gp350
to gp220 may contribute to moving the virus closer to the cell
membrane.
Binding of gp350/220 also triggers capping of CR2 and endocytosis of the virus (38, 60). CR2 can function as a signal
transducer both independently and as part of a signal transduction complex that includes CD19 and CD35 (5). Crosslinking by gp350/220 activates NF-␬B (53, 58) and induces
interleukin-6 via a protein kinase C pathway (4, 62). None of
these signaling events may be critical to penetration of the cell
membrane, but they clearly have potential consequences for
downstream events in infection.
(ii) Epithelial cells. Attachment of the virus to an epithelial
cell is a more-complicated affair (Fig. 2). CR2 is expressed at
least at low levels on some epithelial cells in culture (6) in the
absence of CD19 and CD35, and infection of an epithelial cell
that has been engineered to express high levels of CR2 can be
very efficient (3, 29). Notably, in this regard, EBV-mediated
cross-linking of CR2 on epithelial cells, which lack the other
components of the CR2 signaling complex found on B cells,
stimulates relocalization and clustering of CR2 and the formin
homolog overexpressed in spleen (FHOS/FHOD1). Formins
are molecular scaffolds that nucleate actin, linking signal transduction to actin reorganization and gene transcription (10),
and this may contribute to successful delivery of the virus to
the nucleus of the cell. However, which, or even whether,
epithelial cells normally express CR2 in vivo remains uncertain. Studies with monoclonal antibodies to CR2 were compromised by the finding that the antibody most commonly used
cross-reacted with an unrelated epithelial cell protein (73).
At least three other possible attachment mechanisms have
been proposed that involve neither gp350/220 nor CR2. The
first was a demonstration that virus coated with immunoglobulin A specific to gp350/220 can bind productively to the polymeric immunoglobulin-A receptor (54). This may be particularly relevant to infection via the basolateral surface of an
One of the many conundrums of herpesvirology is why a
herpesvirus is so profligate in its use of four or more envelope
glycoproteins for entry into a cell when other viruses can manage very well with only one or two. Profligacy does, however,
have its advantages. Epstein-Barr virus (EBV), originally recognized for its ability to infect and transform lymphocytes, is
now clearly understood to infect epithelial cells as part of its
normal cycle of persistence in a human host, and under some
circumstances, the virus may infect T cells, natural killer cells,
smooth muscle cells (47), and possibly monocytes as well (11,
50). Our understanding of how EBV enters each of these cell
types is very incomplete, but some of the major players involved in B-cell and epithelial cell infections are being identified, and they provide a window into the flexibility of tropism
that the use of different combinations of virus and cell membrane proteins can provide. This review summarizes what we
know about these players so far.
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J. VIROL.
TABLE 1. EBV envelope glycoproteins with roles in entry into B cells or epithelial cells
Protein/gene
a
gp350/BLLF1
gH/BXLF2
gL/BKRF2
gp42/BZLF2
gB/BALF4
gN/BLRF1
gM/BBRF3
BMRF2
Type
Role in virus entry
Single pass type1
Single pass type 1
Single pass type 2
Single pass type 2
Single pass type 1
Single pass type 1
Multispanning
Multispanning
Attachment to B-cell receptor CR2/CD21
Fusion; attachment to epithelial cell receptor/coreceptor
Chaperone for gH
Fusion; interaction with B-cell coreceptor HLA class II
Fusion
Codependent with gM for expression; possibly involved in postfusion events
Binds integrins; important for infection of polarized epithelial cells
a
EBV envelope proteins are conventionally named for their apparent masses (e.g., gp350); the alphabetical names of homologs in other herpesviruses, if they exist,
(e.g., gH); or their gene names only (e.g., BMRF2).
the virus encounters in vivo, an interaction between a multispan virus membrane protein encoded by the BMRF2 open
reading frame and integrins has been demonstrated (63). Antibodies to integrins and to a BMRF2 fusion protein only
partially block binding to polarized epithelial cells but have a
significant impact on infection via the basolateral surface of the
polarized monolayer. The RGD motif present in an exposed
loop of BMRF2 has been shown to be a ligand for ␤1, ␣5, ␣3,
and ␣v integrins (71). The protein is not required for cell-cell
fusion (12, 31). However, whether the BMRF2 integrin interaction is primarily responsible for attachment or whether it is
most relevant to postattachment events to which signaling may
make an important contribution is not yet clear. There is apparently very little BMRF2 protein in the virion (22).
FUSION AND PENETRATION
Fusion of the EBV envelope with both a B cell and an
epithelial cell requires three glycoproteins, gHgL and gB,
which are conserved throughout the herpesvirus family and
which have been referred to as the core fusion machinery (55).
However, how and where this machinery is activated differs
significantly for each cell type.
(i) B cells. Fusion of the virus with a normal B cell (Fig. 1)
requires endocytosis, which is triggered by the interaction be-
FIG. 1. A putative model of the steps involved in entry of virus into a B lymphocyte. (1) The virus binds to CR2 via gp350, possibly initiating
signaling events and triggering endocytosis. (2) CR2 may now bind to gp220 rather than gp350, and this, combined with the potential flexibility of
CR2, may allow the virus to approach closer to the cell membrane, where gp42 can interact with HLA class II. (3) Interaction of gp42 with HLA
class II triggers the interaction of the core fusion machinery, gHgL and gB, with the endosomal membrane and may also initiate further signaling
events. (4) The virus and endosomal membranes fuse, allowing entry of the tegumented capsid into the cytoplasm. Modified from reference 18,
with permission.
Downloaded from jvi.asm.org by on August 27, 2008
epithelial cell in an immune host, although, since in polarized
cells virus was transported intact from the basolateral to the
apical surface, it may be more relevant to trans epithelial transport than direct infection (9). The second was a demonstration
that in the absence of CR2 a complex of two additional glycoproteins, gH and gL, can serve as epithelial ligands (34, 43).
EBV gH, a type 1 membrane protein, is, like its homologs in
other herpesviruses, misfolded and retained in the endoplasmic reticulum in the absence of the much smaller type 2 protein, gL (27, 72). No separate function in addition to facilitating folding and transport of gH has been ascribed to gL, and
the two proteins, which associate noncovalently, are usually
referred to as a unit, the gHgL complex. EBV derived from a
B cell can bind well to a CR2-negative epithelial cell, but
recombinant viruses that lack gHgL lose this ability (34, 43). A
soluble form of gHgL made in baculovirus can bind specifically
to epithelial cells, but not B cells, and its binding can be
reduced by a monoclonal antibody specific for the gHgL complex (3). The same antibody can also reduce virus binding (34).
These observations have been interpreted to mean that there is
an epithelial cell receptor for gHgL that can serve in attachment. The identity of the molecule is not yet known, but operationally it has been referred to as gHgLR.
Finally, and most intriguing, on polarized epithelial cells,
which presumably resemble more closely the environment that
VOL. 81, 2007
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7827
tween gp350/220 and CR2 and occurs in a low-pH compartment, although low pH itself is not required (32). The virus
proteins that are both necessary and sufficient for efficient
fusion are gHgL, gB, and gp42. A role for gH was first demonstrated when it was shown that virosomes made from proteins depleted of those that bound to an antibody to gH could
attach to B cells but could not fuse (14). It was later confirmed
by analysis of the phenotype of a recombinant gH-null virus
(34). A role for EBV gB was not possible to evaluate genetically, since, unlike its homologs in other herpesviruses, it is
essential for virus morphogenesis and egress (16). However,
when cell-cell fusion assays were developed as models of viruscell fusion, it became apparent that its contribution is also very
important (12, 31).
The fourth and final protein involved in fusion of EBV with
a B cell, gp42, has homologs only among the lymphocryptoviruses (48, 49). It is another type 2 membrane protein, and it
associates noncovalently with gHgL. The original observation
was that gp42 resembles a C-type lectin and that a soluble form
made by replacing the signal sequence with one that is efficiently cleaved can bind to HLA class II (56). Within the
three-part complex of gHgLgp42 in the virus, this interaction is
now thought to provide the trigger for B-cell fusion. A monoclonal antibody that was initially mapped to gH (57) but ultimately proved to react with gp42 (28) inhibited interaction
with HLA class II (27) and also blocked virus-cell fusion (33).
Reciprocally, a monoclonal antibody to HLA class II that
blocked interaction with gp42 also inhibited infection. The
soluble form of gp42 initially used to document HLA class II
binding could inhibit infection by competing with gp42 in the
virus, and B cells that lacked HLA class II could be efficiently
infected only if HLA class II expression was restored (27). A
recombinant gp42-null virus could infect a B cell only if the
cells and the bound virus were treated with an exogenous
fusogen such as polyethylene glycol (65) or if the soluble form
of gp42, which retained a gHgL binding domain at its amino
terminus, was added in trans to reform three-part complexes
(66).
Binding of gp42 to HLA class II shows some allelic specificity in that nonfunctional HLA-DQ alleles have been identified (13), but since all three alleles, HLA-DP, HLA-DQ, and
HLA-DR, can be used, it is perhaps unlikely that this has a
major impact in an outbred human population. A crystal structure of the ectodomain of gp42 liganded to HLA-DR1 has
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FIG. 2. Putative models of the steps involved in entry of virus into epithelial cells after attachment via different cell proteins. (a) Entry via CR2
and gHgLR. (1) The virus binds to CR2 via gp350; signaling may occur, but endocytosis is not triggered. (2) A switch to interaction with gp220,
combined with the potential flexibility of CR2, may allow the virus to approach closer to the cell membrane, where gHgL can interact with gHgLR.
(3) Interaction of gHgL with gHgLR triggers the interaction of the core fusion machinery, gHgL and gB, with the cell membrane. (b) Entry via
gHgLR alone (which is less efficient). (1) There is no separate attachment event. (2) gHgL interacts directly with gHgLR. (3) Interaction of gHgL
with gHgLR triggers the interaction of the core fusion machinery, gHgL and gB, with the cell membrane. (c) Entry via integrins and gHgLR. (1)
BMRF2 interacts with ␣5␤1 integrins, possibly initiating signaling events. (2) gHgL interacts with gHgLR. (3) Interaction of gHgL with gHgLR
triggers the interaction of the core fusion machinery, gHgL and gB, with the cell membrane. Step 4 is the same for all routes: the virus and cell
membrane fuse, allowing entry of the tegumented capsid into the cytoplasm. Modified from reference 18, with permission.
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levels of gB than does B-cell penetration (42), presumably
reflecting a need for more gB in fusion (31). However, the
mechanics of fusion itself remain obscure, as they do for all
herpesviruses. Mutations in an amino-terminal region of gH
that is predicted to form a coiled coil significantly reduced
fusion in a cell-cell fusion assay (44), and insertion or point
mutations in a region close to the transmembrane domain can
either differentially affect B-cell and epithelial cell fusion or
abrogate fusion with either cell type (69, 70). However, some
epithelial cell fusion can be mediated in the absence of gHgL
by a gB construct with a mutation in the cytoplasmic tail. This
has been interpreted to mean that gB may be the critical fusion
protein of EBV. Certainly the crystal structure of the homolog
of gB in herpes simplex virus shows structural homology with
the vesicular stomatitis virus G protein, which is the virus
fusogen (15). Both form trimers characterized by an alphahelical coiled-coil core and extended ␤-hairpins that are characteristic of class I and class II fusion proteins, respectively. At
this point, however, perhaps the most that can be definitively
said is that virus fusion with a B cell requires a trigger that is
transmitted via gp42 to gHgL and gB, whereas fusion with an
epithelial cell requires a trigger that is transmitted directly to
gHgL. Whether there is a temporal cascade of events culminating in fusion mediated by gB or whether gHgL and gB
coordinately contribute to the process remains to be determined.
TRANSFER OF VIRUS BETWEEN B CELLS AND
EPITHELIAL CELLS
gp42 as a switch of tropism. The observation that two-part
gHgL and three-part gHgLgp42 complexes are mutually exclusive for epithelial and B-cell entry suggested that the relative
amounts of each complex in the virion might impact virus
tropism. Analyses of viruses made in B cells and epithelial cells
confirmed this. In a B cell, some three-part gHgLgp42 complexes prove to interact with immature MHC class II in the
endoplasmic reticulum and are targeted to the HLA class II
trafficking pathway, where they are vulnerable to degradation.
This does not happen in an HLA class II-negative epithelial
cell. Epithelial cell viruses are, as a result, rich in complexes
containing gp42 and are as much as two orders of magnitude
more infectious for a B cell than viruses produced by a B cell.
B-cell viruses, lower in three-part complexes but rich in twopart complexes, are better able to infect an epithelial cell,
although the phenotype is not nearly as striking. This suggested
a model (Fig. 3) of persistent infection in which virus reactivating from a latently infected memory B cell as it terminally
differentiates into a plasmablast (23) is equipped to infect an
epithelial cell, whereas virus amplified in an epithelial cell is
strongly B cell tropic (2).
EBV is an orally transmitted virus, and the switch in tropism
effected by levels of gp42, which could impact transmission,
raised the question of the cellular origins of viruses that are
shed in saliva. Viruses made in a B cell, low in gp42, have been
shown to be able to bind to gHgLR on an epithelial cell almost
as well as they can bind CR2 on a B cell. In contrast, virus
made in an epithelial cell, high in gp42, binds gHgLR very
poorly (3). A comparison of the differential abilities of viruses
from saliva and viruses from transformed B cells obtained from
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been solved (37). The protein is most closely related to natural
killer cell receptors, such as the murine receptor LY49, which
interact with major histocompatibility complex (MHC) class I
molecules, but it does not use the canonical surface site which
many C-type lectins use to bind to their ligands. Instead, this
site forms a hydrophobic pocket that, it is suggested, may be
used to accommodate another virus protein or another cell
protein important for virus entry (37), perhaps after triggering
by HLA class II. This is consistent with the observation that
mutations in the hydrophobic pocket do not affect HLA class
II binding but inhibit fusion (52). The contact region on HLADR1 is within the ␤1 domain to one side of the peptide binding
groove. It was thought on theoretical grounds to be likely to
interfere sterically with the binding of T-cell receptors to the
peptide MHC complex and has been shown experimentally to
reduce T-helper-cell recognition of B cells (45, 46, 56). Both
peptide-loaded and immature MHC class II proteins that have
not yet trafficked to the peptide-loading compartment can interact with gp42 (46), and, as discussed below, this has proven
to have an interesting impact on virus tropism. Whether or not
additional signaling events may be initiated as a result of a
gp42-HLA class II interaction is not yet known, but since
signaling via HLA class II molecules is well documented under
other circumstances, this remains a reasonable possibility (67).
(ii) Epithelial cells. Fusion with a normal epithelial cell, at
least one that is nonpolarized, takes place at neutral pH and
does not appear to require endocytosis (3, 32). Epithelial cells
do not constitutively express HLA class II, and thus, not surprisingly, infection is not blocked by the monoclonal antibody
that blocks gp42 and HLA class II interactions. A gp42-null
virus can infect an epithelial cell as well as, or even better than,
wild-type virus. However, not only is gp42 dispensable for
epithelial cell infection, but its presence is actually inhibitory
(66). Stoichiometric analysis of gHgL and gp42 in the virion
indicated that wild-type EBV carries far more gHgL than gp42,
implying that there are two kinds of gH complexes, one the
three-part complex composed of gHgLgp42 and the other a
two-part complex composed of only gHgL. Only three-part
complexes can mediate B-cell infection, but conversion of all
two-part complexes to three-part complexes by the addition of
soluble gp42 in trans blocks epithelial cell infection. This was
interpreted to mean that fusion with an epithelial cell is triggered by a direct interaction between gHgL and a novel epithelial cell surface molecule. Three monoclonal antibodies that
interact with gH or gHgL and have no effect on B-cell infection
can also inhibit infection of epithelial cells, whether virus is
bound via gp350/220 to CR2 or via gHgL to gHgLR. One of
them blocks gHgL binding to gHgLR, suggesting that the cell
molecule that triggers fusion might be the same protein,
gHgLR, which can serve as an attachment receptor in the absence
of CR2. Consistent with this is the observation that soluble
gp42 also blocks binding of the virus to gHgLR. Use of gHgL
for attachment as well as fusion does, however, seem to compromise infection. The virus can bind efficiently to gHgLR on
a CR2-negative epithelial cell, but infection levels are low,
either because fusion becomes less efficient or perhaps because
engagement of gHgLR does not induce a downstream event
required for completion of transport of the virus genome to the
nucleus (3).
Beyond this, epithelial cell penetration also requires higher
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VOL. 81, 2007
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7829
a series of EBV-positive donors then facilitated the determination that the majority of viruses shed in saliva are derived
from HLA class II-negative cells (21). For the most-efficient
transmission, this either means that the virus directly accesses
a B cell in the oral mucosa or that transmission to an epithelial
cell for amplification before accessing a B cell is influenced by
other events. Two have been proposed, and both implicate the
differential use of gp350/220.
gp350/220 as an impediment to epithelial infection. Antibodies to gp350/220, which inhibit infection of a B cell dependent on CR2 for entry, can enhance infection of an epithelial
cell, which is not (64). The effect is not mediated by Fc receptor
binding but is further increased by antibody cross-linking,
which may patch gp350/220 in the virus envelope. Saliva from
EBV-seropositive individuals has similar effects that can be
reversed by depletion of antibodies. The interpretation of
these results was that, if gp350/220 is not absolutely required,
then its presence may impede access of other essential proteins
to the cell surface. Antibodies to gp350/220, an abundant and
immunogenic protein, can overcome this in the saliva of an
immune host to facilitate reinfection of an epithelial cell or to
facilitate transmission through the epithelium in a new host.
There are long-standing observations that epithelial infection is more efficient if epithelial cells are cocultivated with B
cells that are making the virus (19). A novel approach to
investigation of the phenomenon demonstrated that binding
EBV to a B cell before adding both to epithelial cells significantly enhanced infection of the epithelial cells, even in the
absence of internalization and infection of the B cell (51). The
mechanism of this B-cell transfer is uncertain but intriguing.
The authors also suggested that gp350/220 is inhibitory to
direct epithelial cell infection and proposed that the gp350/220
interaction with CR2 on the B cells unmasks envelope components necessary for epithelial cell infection. This parallels
the hypothesis for enhanced infection in the presence of antibodies to gp350/220 in saliva. Neither study addressed what the
envelope components necessary for efficient epithelial cell infection might be, but one likely candidate would be gB, the
protein that is needed in higher amounts for epithelial than for
B cell fusion.
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FIG. 3. Glycoprotein gp42 as a switch of cell tropism. (a) B-cell infection requires an interaction between gHgLgp42 and HLA class II, whereas
epithelial cell infection requires an interaction between gHgL and gHgLR. Complexes lacking gp42 cannot bind to HLA class II, and complexes
containing gp42 cannot bind to gHgLR. (b) gHgLgp42 complexes can interact with HLA class II in the endoplasmic reticulum of a B cell. They
may then be targeted to endosomal vesicles rich in proteases, where incoming proteins are digested into peptides for loading into the peptide
binding groove of HLA class II before continuing on to the cell surface. The result is a loss of gHgLgp42 complexes to degradation and a relative
decrease in gHgLgp42 complexes in viruses produced from B cells or plasmablasts (B-EBV). Complexes in epithelial cells are not lost to this
degradative pathway, and viruses produced from epithelial cells (E-EBV) are richer in gp42. Epithelial cells high in gp42 are better able to infect
B cells, and B cells low in gp42 are better able to infect epithelial cells. Modified from reference 18, with permission.
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J. VIROL.
POSTFUSION EVENTS
SUMMARY
The major players involved in moving EBV through a B-cell
membrane have been identified, at least on the part of the
virus. The model emerging is that gp350/220 binds to CR2,
gp42 binds to HLA class II, and fusion is mediated by gHgL
and gB. The possibility clearly exists, however, that there are
additional cell partners for gHgL and gB. Some of the players
involved in moving EBV through an epithelial cell membrane
have been identified. Fusion involves gHgL and gB, but attachment may involve gp350/220, gHgL, or BMRF2, or possibly, if
the virus is transferred from the surface of a B cell, no attachment receptor specific for any viral protein at all. With the
possible exception of CR2, no cell partners have yet been
identified on an epithelial cell, though there is clear evidence
that they exist. There is also no conceptual understanding of
the mechanism of fusion and very little insight into events
inside the cell that may be triggered by interactions occurring
at the cell surface. Much is still to be learned.
Despite the incompleteness of our understanding, however,
what we know already provides a fascinating glimpse into differential use of virus proteins and cell partners to manipulate
virus tropism and influence virus trafficking between one cell
compartment and another. EBV is associated with human malignancies, and primary infection in a minority of individuals
causes acute, but self-limiting, mononucleosis. The majority of
humans, however, live without incident with a virus that con-
ACKNOWLEDGMENTS
Research by L.M.H.-F. is supported by Public Health Service grants
AI061017 from the National Institute of Allergy and Infectious Diseases and DE016669 from the National Institute of Dental and Craniofacial Research.
REFERENCES
1. Beisel, C., J. Tanner, T. Matsuo, D. Thorley-Lawson, F. Kezdy, and E. Kieff.
1985. Two major outer envelope glycoproteins of Epstein-Barr virus are
encoded by the same gene. J. Virol. 54:665–674.
2. Borza, C. M., and L. M. Hutt-Fletcher. 2002. Alternate replication in B cells
and epithelial cells switches tropism of Epstein-Barr virus. Nat. Med. 8:594–
599.
3. Borza, C. M., A. J. Morgan, S. M. Turk, and L. M. Hutt-Fletcher. 2004. Use
of gHgL for attachment of Epstein-Barr virus to epithelial cells compromises
infection. J. Virol. 78:5007–5014.
4. D’Addario, M., T. A. Libermann, J. Xu, A. Ahmad, and J. Menezes. 2001.
Epstein-Barr virus and its glycoprotein-350 upregulate IL-6 in human B cells
via CD21, involving activation of NF-kB and different signaling pathways. J.
Mol. Biol. 308:501–514.
5. Fearon, D. T., and R. H. Carter. 1995. The CD19/CR2/TAPA-1 complex of
B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol.
13:127–149.
6. Fingeroth, J. D., M. E. Diamond, D. R. Sage, J. Hayman, and J. L. Yates.
1999. CD-21 dependent infection of an epithelial cell line, 293, by EpsteinBarr virus. J. Virol. 73:2115–2125.
7. Fingeroth, J. D., J. J. Weis, T. F. Tedder, J. L. Strominger, P. A. Biro, and
D. T. Fearon. 1984. Epstein-Barr virus receptor of human B lymphocytes is
the C3d complement CR2. Proc. Natl. Acad. Sci. USA 81:4510–4516.
8. Frade, R., M. Barel, B. Ehlin-Henricksson, and G. Klein. 1985. Gp140, the
C3d receptor of human B lymphocytes, is also the Epstein-Barr virus receptor. Proc. Natl. Acad. Sci. USA 82:1490–1493.
9. Gan, Y., J. Chodosh, A. Morgan, and J. W. Sixbey. 1997. Epithelial cell
polarization is a determinant in the infectious outcome of immunoglobulin
A-mediated entry by Epstein-Barr virus. J. Virol. 71:519–526.
10. Gill, M. B., J. Roecklein-Canfield, D. R. Sage, M. Zambela-Soediono, N.
Longtine, M. Uknis, and J. D. Fingeroth. 2004. EBV attachment stimulates
FHOF/FHOD1 redistribution and co-aggregation with CD21:formin interactins with the cytoplasmic domain of human CD21. J. Cell Sci. 117:2709–
2720.
11. Guerreiro-Cacais, A. O., L. Li, D. D. Donat, M. T. Bejarano, A. J. Morgan,
M. Masucci, L. M. Hutt-Fletcher, and V. Levitsky. 2004. The capacity of
Epstein-Barr virus to infect monocytes and inhibit their development into
dendritic cells is affected by the cell type supporting virus replication. J. Gen.
Virol. 85:2767–2778.
12. Haan, K. M., S. K. Lee, and R. Longnecker. 2001. Different functional
domains in the cytoplasmic tail of glycoprotein gB are involved in EpsteinBarr virus-induced membrane fusion. Virology 290:106–114.
13. Haan, K. M., and R. Longnecker. 2000. Coreceptor restriction within the
HLA-DQ locus for Epstein-Barr virus infection. Proc. Natl. Acad. Sci. USA
97:9252–9257.
14. Haddad, R. S., and L. M. Hutt-Fletcher. 1989. Depletion of glycoprotein
gp85 from virosomes made with Epstein-Barr virus proteins abolishes their
ability to fuse with virus receptor-bearing cells. J. Virol. 63:4998–5005.
15. Heldwein, E. E., H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and
S. C. Harrison. 2006. Crystal structure of glycoprotein B from herpes simplex
virus 1. Science 313:217–220.
16. Herrold, R. E., A. Marchini, S. Frueling, and R. Longnecker. 1995. Glycoprotein 110, the Epstein-Barr virus homolog of herpes simplex virus glycoprotein B, is essential for Epstein-Barr virus replication in vivo. J. Virol.
70:2049–2054.
17. Hummel, M., D. Thorley-Lawson, and E. Kieff. 1984. An Epstein-Barr virus
DNA fragment encodes messages for the two major envelope glycoproteins
(gp350/300 and gp220/200). J. Virol. 49:413–417.
18. Hutt-Fletcher, L. M. 2005. EBV entry and epithelial infection, p. 359–378. In
E. S. Robertson (ed.), Epstein-Barr virus. Caister Academic Press, Norfolk,
England.
19. Imai, S., J. Nishikawa, and K. Takada. 1998. Cell-to-cell contact as an
efficient mode of Epstein-Barr virus infection of diverse human epithelial
cells. J. Virol. 72:4371–4378.
20. Janz, A., M. Oezel, C. Kurzeder, J. Mautner, D. Pich, M. Kost, W. Hammerschmidt, and H. J. Delecluse. 2000. Infectious Epstein-Barr virus lacking
major glycoprotein BLLF1 (gp350/220) demonstrates the existence of additional viral ligands. J. Virol. 74:10142–10152.
21. Jiang, R., R. S. Scott, and L. M. Hutt-Fletcher. 2006. Epstein-Barr virus shed
in saliva is high in B-cell-tropic gp42. J. Virol. 80:7281–7283.
Downloaded from jvi.asm.org by on August 27, 2008
There is regrettably very little concrete information about
what happens between fusion and the arrival of the virus genome in the nucleus, only speculation based on what is known
for other herpesviruses. Allusion has been made throughout
this brief review to the likelihood that signaling events triggered by interactions between the virus envelope and cell proteins have important consequences for postfusion events, but
how they may impact intracellular transport and uncoating of
the virion is a subject that has not yet been substantively addressed. Two glycoproteins in the virus envelope have, however, also been tentatively implicated in what are presumably
immediate postfusion events that may perhaps have no connection to cell signaling. These are glycoprotein gN, a small
type I membrane protein, and glycoprotein gM. Glycoprotein
gM is a multispan membrane protein with a long, highly
charged cytoplasmic tail rich in prolines and replete with potential phosphorylation sites that might provide a mechanism
for the regulation of protein-protein interactions. Glycoproteins gN and gM are codependent for expression (24, 25) and
play important roles in virus assembly and acquisition of the
final virus envelope. Only very little enveloped virus is made by
a recombinant EBV that lacks gN and gM. However, that
enveloped virus which is made and is able to bind specifically
to CR2 on a B cell is impaired in infection in a way that cannot
be rescued by addition of exogenous fusogens such as polyethylene glycol. One hypothesis is that a complex that is necessary
for assembly of an enveloped particle when a tegumented
capsid associates with a membrane is also essential to its disassembly when a tegumented capsid must disassociate from a
membrane with which its envelope has fused.
tinually infects their lymphocytes and epithelial cells. The strategies that have evolved to make this possible also continue to
be remarkable.
VOL. 81, 2007
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
7831
actions by Epstein-Barr virus gp42 results in reduced T helper cell recognition. Proc. Natl. Acad. Sci. USA 100:11583–11588.
Ressing, M. E., D. van Leeuwen, F. A. W. Verreck, S. Keating, R. Gomez,
K. L. M. C. Franken, T. H. M. Ottenhoff, M. Spriggs, T. N. Schumacher,
L. M. Hutt-Fletcher, M. Rowe, and E. J. H. J. Wiertz. 2005. Epstein-Barr
virus gp42 is posttranslationally modified to produce soluble gp42 that mediates class II immune evasion. J. Virol. 79:841–852.
Rickinson, A. B., and E. Kieff. 2007. Epstein-Barr virus, p. 2655–2700. In
D. M. Knipe and P. M. Howley (ed.), Fields virology. Lippincott Williams &
Wilkins, Philadelphia, PA.
Rivailler, P., Y.-G. Cho, and F. Wang. 2002. Complete genomic sequence of
an Epstein-Barr virus-related herpesvirus naturally infecting a New World
primate: a defining point in the evolution of oncogenic lymphocryptoviruses.
J. Virol. 76:12055–12068.
Rivailler, P., H. Jiang, Y.-G. Cho, C. Quink, and F. Wang. 2002. Complete
nucleotide sequence of the rhesus lymphocryptovirus: genetic validation for
an Epstein-Barr virus animal model. J. Virol. 76:421–426.
Savard, M., C. Belanger, M. Tardif, P. Gourde, L. Flamand, and J. Gosselin.
2000. Infection of primary human monocytes by Epstein-Barr virus. J. Virol.
74:2612–2619.
Shannon-Lowe, C. D., B. Neuhierl, G. Baldwin, A. B. Rickinson, and H.-J.
Delecluse. 2006. Resting B cells as a transfer vehicle for Epstein-Barr
virus infection of epithelial cells. Proc. Natl. Acad. Sci. USA 103:7065–
7070.
Silva, A. L., J. Omerovic, T. S. Jardetzky, and R. Longnecker. 2004. Mutational analysis of Epstein-Barr virus glycoprotein gp42 reveals functional
domains not involved in receptor binding but required for membrane fusion.
J. Virol. 78:5946–5956.
Sinclair, A. J., and P. J. Farrell. 1995. Host cell requirements for efficient
infection of quiescent primary B lymphocytes by Epstein-Barr virus. J. Virol.
69:5461–5468.
Sixbey, J. W., and Q.-Y. Yao. 1992. Immunoglobulin A-induced shift of
Epstein-Barr virus tissue tropism. Science 255:1578–1580.
Spear, P. G., and R. Longnecker. 2003. Herpesvirus entry: an update. J. Virol. 77:10179–10185.
Spriggs, M. K., R. J. Armitage, M. R. Comeau, L. Strockbine, T. Farrah,
B. MacDuff, D. Ulrich, M. R. Alderson, J. Müllberg, and J. I. Cohen.
1996. The extracellular domain of the Epstein-Barr virus BZLF2 protein
binds the HLA-DR ␤ chain and inhibits antigen presentation. J. Virol.
70:5557–5563.
Strnad, B. C., T. Schuster, R. Klein, R. F. Hopkins III, T. Witmer, R. H.
Neubauer, and H. Rabin. 1982. Production and characterization of monoclonal antibodies against the Epstein-Barr virus membrane antigen. J. Virol.
41:258–264.
Sugano, N., W. Chen, M. L. Roberts, and N. R. Cooper. 1997. Epstein-Barr
virus binding to CD21 activates the initial viral promoter via NF-␬B induction. J. Exp. Med. 186:731–737.
Szakonyi, G., M. G. Klein, J. P. Hannan, K. A. Young, R. Z. Ma, R. Asokan,
V. M. Holers, and X. S. Chen. 2006. Structure of the Epstein-Barr virus
major envelope glycoprotein. Nat. Struct. Mol. Biol. 13:996–1001.
Tanner, J., J. Weis, D. Fearon, Y. Whang, and E. Kieff. 1987. Epstein-Barr
virus gp350/220 binding to the B lymphocyte C3d receptor mediates adsorption, capping, and endocytosis. Cell 50:203–213.
Tanner, J., Y. Whang, J. Sample, A. Sears, and E. Keiff. 1988. Soluble
gp350/220 and deletion mutant glycoproteins block Epstein-Barr virus adsorption to lymphocytes. J. Virol. 62:4452–4464.
Tanner, J. E., C. Alfieri, T. A. Chatila, and F. Diaz-Mitoma. 1996. Induction
of interleukin-6 after stimulation of human B-cell CD21 by Epstein-Barr
virus glycoproteins gp350 and gp220. J. Virol. 70:570–575.
Tugizov, S. M., J. W. Berline, and J. M. Palefsky. 2003. Epstein-Barr virus
infection of polarized tongue and nasopharyngeal epithelial cells. Nat. Med.
9:307–314.
Turk, S. M., R. Jiang, L. S. Chesnokova, and L. M. Hutt-Fletcher. 2006.
Antibodies to gp350/220 enhance the ability of Epstein-Barr virus to infect
epithelial cells. J. Virol. 80:9628–9633.
Wang, X., and L. M. Hutt-Fletcher. 1998. Epstein-Barr virus lacking
glycoprotein gp42 can bind to B cells but is not able to infect. J. Virol.
72:158–163.
Wang, X., W. J. Kenyon, Q. X. Li, J. Mullberg, and L. M. Hutt-Fletcher.
1998. Epstein-Barr virus uses different complexes of glycoproteins gH and gL
to infect B lymphocytes and epithelial cells. J. Virol. 72:5552–5558.
Watt, T. H. 1997. Signaling via MHC class II molecules, p. 141–161. In M.
Harnete and K. P. Rigley (ed.), Lymphocyte signaling: mechanisms, subversion and manipulation. John Wiley and Sons Ltd., New York, NY.
Weisman, H. F., T. Bartow, M. K. Leppo, H. C. Marsh, Jr., G. R. Carson,
M. F. Concino, M. P. Boyle, K. H. Roux, M. L. Weisdeldt, and D. T. Fearon.
1990. Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis.
Science 249:146–151.
Wu, L., C. M. Borza, and L. M. Hutt-Fletcher. 2005. Mutations of EpsteinBarr virus gH that are differentially able to support fusion with B cells or
epithelial cells. J. Virol. 79:10923–10930.
Downloaded from jvi.asm.org by on August 27, 2008
22. Johannsen, E., M. Luftig, M. R. Chase, S. Weicksel, E. Cahir-McFarland, D.
Illanes, D. Sarracino, and E. Kieff. 2004. Proteins of purified Epstein-Barr
virus. Proc. Natl. Acad. Sci. USA 101:16286–16291.
23. Laichalk, L. L., and D. A. Thorley-Lawson. 2005. Terminal differentiation
into plasma cells initiates the replicative cycle of Epstein-Barr virus in vivo.
J. Virol. 79:1296–1307.
24. Lake, C. M., and L. M. Hutt-Fletcher. 2000. Epstein-Barr virus that lacks
glycoprotein gN is impaired in assembly and infection. J. Virol. 74:11162–
11172.
25. Lake, C. M., S. J. Molesworth, and L. M. Hutt-Fletcher. 1998. The EpsteinBarr virus (EBV) gN homolog BLRF1 encodes a 15-kilodalton glycoprotein
that cannot be authentically processed unless it is coexpressed with the EBV
gM homolog BBRF3. J. Virol. 72:5559–5564.
26. Lambris, J. D., V. S. Ganu, S. Hirani, and H. J. Muller-Eberhard. 1985.
Mapping of the C3d receptor (CR2) binding site and a neoantigenic site in
the C3d domain of the third component of complement. Proc. Natl. Acad.
Sci. USA 82:4235–4239.
27. Li, Q. X., M. K. Spriggs, S. Kovats, S. M. Turk, M. R. Comeau, B. Nepom,
and L. M. Hutt-Fletcher. 1997. Epstein-Barr virus uses HLA class II as a
cofactor for infection of B lymphocytes. J. Virol. 71:4657–4662.
28. Li, Q. X., S. M. Turk, and L. M. Hutt-Fletcher. 1995. The Epstein-Barr virus
(EBV) BZLF2 gene product associates with the gH and gL homologs of
EBV and carries an epitope critical to infection of B cells but not of epithelial cells. J. Virol. 69:3987–3994.
29. Li, Q. X., L. S. Young, G. Niedobitek, C. W. Dawson, M. Birkenbach, F.
Wang, and A. B. Rickinson. 1992. Epstein-Barr virus infection and replication in a human epithelial system. Nature 356:347–350.
30. Martin, D. R., A. Yuryev, K. R. Kalli, D. T. Fearon, and J. M. Ahearn.
1991. Determination of the structural basis for selective binding of Epstein-Barr virus to human complement receptor type 2. J. Exp. Med.
174:1299–1311.
31. McShane, M. P., and R. Longnecker. 2004. Cell-surface expression of a
mutated Epstein-Barr virus glycoprotein B allows fusion independent of
other viral proteins. Proc. Natl. Acad. Sci. USA 101:17474–17479.
32. Miller, N., and L. M. Hutt-Fletcher. 1992. Epstein-Barr virus enters B cells
and epithelial cells by different routes. J. Virol. 66:3409–3414.
33. Miller, N., and L. M. Hutt-Fletcher. 1988. A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus. J. Virol. 62:2366–2372.
34. Molesworth, S. J., C. M. Lake, C. M. Borza, S. M. Turk, and L. M. HuttFletcher. 2000. Epstein-Barr virus gH is essential for penetration of B cells
but also plays a role in attachment of virus to epithelial cells. J. Virol.
74:6324–6332.
35. Moore, M. D., M. J. Cannon, A. Sewall, M. Finlayson, M. Okimoto, and
G. R. Nemerow. 1991. Inhibition of Epstein-Barr virus infection in vitro and
in vivo by soluble CR2 (CD21) containing two short consensus repeats.
J. Virol. 65:3559–3565.
36. Moore, M. D., R. G. DiScipio, N. R. Cooper, and G. R. Nemerow. 1989.
Hydrodynamic, electron microscopic, and ligand-binding analysis of the
Epstein-Barr virus/C3dg receptor (CR2). J. Biol. Chem. 34:20576–20582.
37. Mullen, M. M., K. M. Haan, R. Longnecker, and T. S. Jardetzky. 2002.
Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II
receptor HLA-DR1. Mol. Cell 9:375–385.
38. Nemerow, G. R., and N. R. Cooper. 1984. Early events in the infection of
human B lymphocytes by Epstein-Barr virus. Virology 132:186–198.
39. Nemerow, G. R., R. A. Houghton, M. D. Moore, and N. R. Cooper. 1989.
Identification of the epitope in the major envelope proteins of Epstein-Barr
virus that mediates viral binding to the B lymphocyte EBV receptor (CR2).
Cell 56:369–377.
40. Nemerow, G. R., C. Mold, V. K. Schwend, V. Tollefson, and N. R. Cooper.
1987. Identification of gp350 as the viral glycoprotein mediating attachment
of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence
homology of gp350 and C3 complement fragment C3d. J. Virol. 61:1416–
1420.
41. Nemerow, G. R., R. Wolfert, M. McNaughton, and N. R. Cooper. 1985.
Identification and characterization of the Epstein-Barr virus receptor on
human B lymphocytes and its relationship to the C3d complement receptor
(CR2). J. Virol. 55:347–351.
42. Neuhierl, B., R. Feederle, W. Hammerschmidt, and H. J. Delecluse. 2002.
Glycoprotein gp110 of Epstein-Barr virus determines viral tropism and efficiency of infection. Proc. Natl. Acad. Sci. USA 99:15036–15041.
43. Oda, T., S. Imai, S. Chiba, and K. Takada. 2000. Epstein-Barr virus lacking
glycoprotein gp85 cannot infect B cells and epithelial cells. Virology 276:
52–58.
44. Omerovic, J., L. Lev, and R. Longnecker. 2005. The amino terminus of
Epstein-Barr virus glycoprotein gH is important for fusion with B cells and
epithelial cells. J. Virol. 79:12408–12415.
45. Ressing, M. E., D. van Leeuwen, F. A. W. Verreck, R. Gomez, B. Heemskerk,
M. Toebes, M. M. Mullen, T. S. Jardetzky, R. Longnecker, M. W. Schilham,
T. H. M. Ottenhoff, J. Neefjes, T. N. Schumacher, L. M. Hutt-Fletcher, and
E. J. H. J. Wiertz. 2003. Interference with T cell receptor-HLA-DR inter-
MINIREVIEW
7832
MINIREVIEW
70. Wu, L., and L. M. Hutt-Fletcher. 20 February 2007. Point mutations in EBV
gH that abrogate or differentially affect B cell and epithelial cell fusion.
Virology 363:148–155. [Epub ahead of print.]
71. Xiao, J., J. M. Palefsky, R. Herrera, and S. M. Tugizov. 2007. Characterization of the Epstein-Barr virus glycoprotein BMRF2. Virology 359:
382–396.
72. Yaswen, L. R., E. B. Stephens, L. C. Davenport, and L. M. Hutt-Fletcher.
J. VIROL.
1993. Epstein-Barr virus glycoprotein gp85 associates with the BKRF2 gene
product and is incompletely processed as a recombinant protein. Virology
195:387–396.
73. Young, L. S., C. W. Dawson, K. W. Brown, and A. B. Rickinson. 1989.
Identification of a human epithelial cell surface protein sharing an epitope
with the C3d/Epstein-Barr virus receptor molecule of B lymphocytes. Int. J.
Cancer 43:786–794.
Downloaded from jvi.asm.org by on August 27, 2008