CD21-Dependent infection of an epithelial cell line, 293, by Epstein-Barr virus.

Abstract

Epstein-Barr virus (EBV) is invariably present in undifferentiated nasopharyngeal carcinomas, is found sporadically in other carcinomas, and replicates in the differentiated layer of the tongue epithelium in lesions of oral hairy leukoplakia. However, it is not clear how frequently or by what mechanism EBV infects epithelial cells normally. Here, we report that a human epithelial cell line, 293, can be stably infected by EBV that has been genetically marked with a selectable gene. We show that 293 cells express a relatively low level of CD21, that binding of fluorescein-labeled EBV to 293 cells can be detected, and that both the binding of virus to cells and infection can be blocked with antibodies specific for CD21. Two proteins known to form complexes with CD21 on the surface of lymphoid cells, CD35 and CD19, could not be detected at the surface of 293 cells. All infected clones of 293 cells exhibited tight latency with a pattern of gene expression similar to that of type II latency, but productive EBV replication and release of infectious virus could be induced inefficiently by forced expression of the lytic transactivators, R and Z. Low levels of mRNA specific for the transforming membrane protein of EBV, LMP-1, as well as for LMP-2, were detected; however, LMP-1 protein was either undetectable or near the limit of detection at less than 5% of the level typical of EBV-transformed B cells. A slight increase in expression of the receptor for epidermal growth factor, which can be induced in epithelial cells by LMP-1, was detected at the cell surface with two EBV-infected 293 cell clones. These results show that low levels of surface CD21 can support infection of an epithelial cell line by EBV. The results also raise the possibility that in a normal infection of epithelial cells by EBV, the LMP-1 protein is not expressed at levels that are high enough to be oncogenic and that there might be differences in the cells of EBV-associated epithelial cancers that have arisen to allow for elevated expression of LMP-1.

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JOURNAL OF VIROLOGY,
0022-538X/99/$04.00?0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Mar. 1999, p. 2115–2125 Vol. 73, No. 3
CD21-Dependent Infection of an Epithelial Cell Line, 293, by
Epstein-Barr Virus
JOYCE D. FINGEROTH,1MARGARET E. DIAMOND,2DAVID R. SAGE,1JODY HAYMAN,2
AND JOHN L. YATES2*
Divisions of Infectious Disease and Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard
Medical School, Boston, Massachusetts 02215,1and Department of Genetics, Roswell Park Cancer Institute,
Buffalo, New York 142632
Received 10 September 1998/Accepted 13 November 1998
Epstein-Barr virus (EBV) is invariably present in undifferentiated nasopharyngeal carcinomas, is found
sporadically in other carcinomas, and replicates in the differentiated layer of the tongue epithelium in lesions
of oral hairy leukoplakia. However, it is not clear how frequently or by what mechanism EBV infects epithelial
cells normally. Here, we report that a human epithelial cell line, 293, can be stably infected by EBV that has
been genetically marked with a selectable gene. We show that 293 cells express a relatively low level of CD21,
that binding of fluorescein-labeled EBV to 293 cells can be detected, and that both the binding of virus to cells
and infection can be blocked with antibodies specific for CD21. Two proteins known to form complexes with
CD21 on the surface of lymphoid cells, CD35 and CD19, could not be detected at the surface of 293 cells. All
infected clones of 293 cells exhibited tight latency with a pattern of gene expression similar to that of type II
latency, but productive EBV replication and release of infectious virus could be induced inefficiently by forced
expression of the lytic transactivators, R and Z. Low levels of mRNA specific for the transforming membrane
protein of EBV, LMP-1, as well as for LMP-2, were detected; however, LMP-1 protein was either undetectable
or near the limit of detection at less than 5% of the level typical of EBV-transformed B cells. A slight increase
in expression of the receptor for epidermal growth factor, which can be induced in epithelial cells by LMP-1,
was detected at the cell surface with two EBV-infected 293 cell clones. These results show that low levels of
surface CD21 can support infection of an epithelial cell line by EBV. The results also raise the possibility that
in a normal infection of epithelial cells by EBV, the LMP-1 protein is not expressed at levels that are high
enough to be oncogenic and that there might be differences in the cells of EBV-associated epithelial cancers
that have arisen to allow for elevated expression of LMP-1.
Accumulating evidence indicates that a typical infection of a
person by Epstein-Barr virus (EBV) is primarily an infection of
the person’s B cells, both during the acute phase of infection
(1, 20, 38) and during life-long latency (34, 35, 42, 49). EBV
readily infects human B cells in vitro, by attaching to CD21 at
the cell surface, and establishes a latent infection which trans-
forms the B cells into proliferating lymphoblasts (21, 22). It is
clear that at some frequency EBV infects nonlymphoid cell
types in vivo, since its genomes can be found in a variety of
nonlymphoid cancers, primarily epithelial and, most notably,
undifferentiated nasopharyngeal carcinoma (NPC) (39). In pa-
tient with AIDS, EBV can cause oral hairy leukoplakia, an
active EBV infection of the differentiated epithelium of the
tongue (15, 56). Very little is known about how frequently
EBV infects epithelial cells during normal human infection,
about how the virus gains entry into epithelial cells, or whether
such an infection typically becomes latent, becomes lytic, or is
aborted.
Studies of the infection of epithelial cells by EBV have been
limited because EBV does not readily infect epithelial cell lines
in culture. The EBV receptor for B cells is CD21, or comple-
ment receptor 2 (CR2), which serves as the receptor for com-
plement component C3d,g. EBV binding to CD21 is effected
by a viral envelope protein, gp350/220, which shares a region of
sequence similarity with C3d,g (8, 10, 36). Expression of CD21
at high levels in epithelial cells from a stably transfected cDNA
was shown to be capable of mediating efficient attachment of
EBV to epithelial cells, which led to a transient infection (30).
Two human epithelial cell lines, RHEK and HeLa, were shown
to express very low levels of CD21 and/or its mRNA and were
able to bind EBV at the cell surface, but EBV binding was not
shown to be dependent on CD21 (3). Since these cell lines
were not shown to become infected by EBV, it has not been
clear whether such low levels of surface CD21 would be suffi-
cient to support uptake of EBV by these cells. The detection of
CD21 by monoclonal antibodies (MAbs) on epithelial surfaces
of tissue sections has been called into question (3), and un-
equivocal evidence has not been obtained to substantiate the
presence of CD21 on epithelial cells in vivo. Recent studies in
vitro suggest certain epithelial cell lines that do not express
CR2 can be infected by EBV if they are cocultivated with
virus-releasing cells (19). Cell lines that express the immuno-
globulin A (IgA) receptor, SC, are susceptible to uptake of
EBV mediated by EBV-specific IgA, suggesting a plausible
mechanism for the infection of mucosal epithelial cells in in-
dividuals who have acquired immunity to EBV (47).
Here we report using recombinant EBV carrying a G418
resistance gene to test whether epithelial cell lines could be
stably infected by EBV to generate drug-resistant cell clones,
an approach that allows detection of rare, stably infected cells
in a tested population. One cell line among several that were
tested, a human embryonic kidney cell line, 293, could be stably
infected by EBV at a low efficiency. Our studies indicate that
infection of 293 cells by EBV is mediated by CD21. Recently,
Takada and coworkers reported using a similar approach to
* Corresponding author. Mailing address: Department of Genetics,
Roswell Park Cancer Institute, Elm and Carlton St., Buffalo, NY
14263. Phone: (716) 845-8964. Fax: (716) 845-8449. E-mail: yates
@sc3101.med.buffalo.edu.
2115

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detect stable infection of cell lines derived from human gastric
carcinomas by soluble EBV and infection of several other
epithelial cell lines with cell-associated virus, but in contrast to
our findings, infection of these cell lines did not appear to be
mediated by CD21 (19, 55).
Little is known about how EBV gene expression is con-
trolled in epithelial cells. The issue is relevant to the involve-
ment of EBV in NPC, where the EBV transforming membrane
protein, LMP-1, is suspected of playing a major role (23, 40).
In contrast to the readily detectable expression of LMP-1 in
NPC, LMP-1 protein was usually undetectable or near the
detection limit in this study and in previous studies of experi-
mentally infected epithelial cell lines (19, 24, 55). This finding
suggests that one aspect of NPC may be the abnormal expres-
sion of LMP-1. In NPC, the LMP-1 gene is transcribed from
two promoters, giving rise to a 3.5-kb mRNA as well as to the
more abundant 2.8-kb mRNA, the form which is expressed
almost exclusively in B cells (44). In infected 293 cells, only a
3.5-kb LMP-1 mRNA was detected, at low abundance.
MATERIALS AND METHODS
Viruses and cell lines. 293 (13) and RHEK (43) are cell lines that were derived
from human embryonic kidney epithelium by transformation with human ade-
novirus DNA (293) or an adenovirus–simian virus 40 hybrid (RHEK). 293 cells
are available from American Type Culture Collection (ATCC), Manassas, Va.;
RHEK cells were kindly provided by J. Rhim, Harvard University. P3-531 virus
stocks were obtained by transfecting P3HR1 clone 16 cells (16) with p531, a
plasmid carrying EBV positions 43935 to 62280 of strain B95-8, with a CMVIE-
neo gene (neo gene driven by the cytomegalovirus immediate-early promoter)
replacing the BHRF1 open reading frame, as previously described (27). B-652 is
a derivative of B95-8 virus that carries the CMVIE-neo gene inserted down-
stream of the EBNA1 coding exon along with a duplication of a few hundred
base pairs in a manner that avoided disrupting any EBV gene or known tran-
scription unit. A clone of B95-8 cells carrying recombinant B-652 was isolated
and used to generate stocks of virus containing about equal amounts of B-652
and parental B95-8 viral genomes (27a). Adherent cell lines were cultured in
Iscove’s modified Dulbecco’s medium; B-cell lines were cultured in RPMI 1640
medium containing 9% fetal bovine serum and the antibiotics penicillin and
ampicillin (all components from Gibco BRL).
Stable infection with recombinant EBV. The EBV-infected clones of 293 that
were characterized in most detail were obtained by cocultivating gamma-irradi-
ated electroporated P3HR1 cells with 293 cells. For all other infections, virus
stocks that had been passed through 0.2-?m-pore-size filters were used. Prior to
infection, 293 cells were trypsinized from cultures that had just reached conflu-
ence and replated at approximately 1:10, usually into six-well culture dishes. The
next day, culture medium was removed and fresh medium was added along with
dilutions of recombinant virus stock. Two days later, the growth medium was
replaced with fresh medium containing G418 (Gibco BRL) at 700 ?g/ml (active
drug concentration), which was replenished as needed until G418-resistant
(G418r) colonies were counted 2 to 3 weeks after infection. To determine titers
for Burkitt’s lymphoma line Raji or BL30, cells were infected in 48-well plates
containing 25,000 cells and 0.4 ml medium in each well. Different dilutions of
virus stock were added to the wells, typically using 24 wells for each dilution. Two
days later, approximately half of the medium in each well was removed by gentle
aspiration and replaced with fresh medium containing sufficient G418 to achieve
active drug concentrations of 840 and 1,120 ?g/ml for Raji and BL30, respec-
tively. Medium was replaced periodically as it became acidified, and wells were
scored for emergent clones until 3 to 4 weeks after infection. G418rvirus titers
were determined by using Poisson statistics.
Blocking infection with antibodies. 293 cells were seeded into six-well plates
from just-confluent cultures, using a 1:4 split for use the next day. Anti-CD21 or
control antibodies were added to each well at their working concentrations,
determined as described below, along with 0.4 ml of medium, and the plates were
sealed with Parafilm and rocked for 30 min at 24°C; 30 ?l of virus stock was
added, and the plates were sealed and rocked at 24°C for 1 h. The medium with
virus was then removed, and the cells were rinsed twice with medium and
returned to culture. Alternatively, after incubation with the first antibody, the
medium with antibody was removed, the cells were rinsed once, and cells were
incubated with a secondary (cross-linking) antibody for 30 min at 24°C before
virus was added. The following day, cells in each well were removed by using
trypsin, washed free of trypsin, and replated into three wells of a six-well plate.
Beginning the second day after infection, the cells were cultured in medium
containing 740 ?g of G418 (active) per ml, and viable clones were counted 12
days later. The EBV strain used for this experiment, P3-?DS-42, will be de-
scribed elsewhere. It is a P3HR1 derivative that has the same selective marker
insertion as P3-531, and was produced in B95-8 cells at a high titer.
EBV labeling and binding to cells. Purification of EBV, coupling to fluorescein
isothiocyanate (FITC), and EBV binding and binding inhibition assays were
essentially as described elsewhere (8), except that in some experiments EBV was
coupled to FITC (isomer I; Sigma, St. Louis, Mo.) that had been dissolved in
dimethyl sulfoxide at a concentration of 5 mg/ml and incubated 1:100 with
purified virus in carbonate-bicarbonate buffer.
Flow cytometry. For cytometric analysis, we used 1 ? 105to 5 ? 105cells per
sample. Adherent cells were suspended by incubation in 20 mM EDTA min
phosphate-buffered saline (without calcium and magnesium) at 37°C for 10 min.
Adherent cells or cells grown in suspension culture were then washed twice with
fresh culture medium containing 2% heat-inactivated fetal bovine serum. Cells
were incubated with antibody for 3 min on ice, washed with phosphate-buffered
saline, incubated with a secondary antibody for another 30 min on ice, and then
washed two additional times. Propidium iodide was added to give a final con-
centration of 5 ?g/ml. Live cells were analyzed by flow cytometry on a FACScan
(Becton Dickinson). The histograms presented in Fig. 1A, 2, 3 and 8 are repre-
sentative of independent analyses that were performed two to seven times.
Antibodies. (i) For cell surface antigens: Mouse MAbs were as follows: HB-5
(anti-CD21) and 543 (anti-CD35), obtained from the ATCC; OKB7 (anti-
CD21), purchased from Ortho Diagnostics, Raritan, N.J.; B-C3 (anti-CD19),
from Biosource, Camarillo, Calif.; 5A6 (anti-CD81, TAPA-1) a gift of Shoshana
Levy, Stanford University School of Medicine, Stanford, Calif.; anti-Leu-13, a
gift from Ron Evans and Sharon Evans, Roswell Park Cancer Institute, Buffalo,
N.Y.; and LB3.1 (anti-HLA class II) and W6/32 (anti-HLA class I), gifts from
Jack Strominger, Harvard University, Cambridge, Mass. Each antibody was
titered and used for cell staining or for blocking experiments at a concentration
that was 10-fold in excess of saturation based on cytometric analysis using a
standard lymphoblastoid cell line such as X50-7 (or, in the case of CD35,
THP-1). Anti-epidermal growth factor receptor (EGFR) (Ab-1, clone 528; On-
cogene Sciences, Cambridge, Mass.) was used at 10 ?g/ml.
(ii) Control MAb. UPC10, an IgG2a mouse MAb with hapten specificity for
?-2-6-linked fructosan, was purchased from Cappel-Organon Teknika, Durham,
N.C. MOPC21, an IgG2b mouse MAb, was purchased from Cappel; P3, an IgG1
MAb was from the ATCC. UPC10, MOPC21, and P3 have no known specificity
for human cells. They were used as isotype controls at a concentration of 10
?g/ml unless stated otherwise.
(iii) Rabbit antisera. Rabbit anti-human CD21 was prepared as described
elsewhere (7). Preimmune or normal rabbit serum was used as a control. Rabbit
sera were used at a dilution of 1:200. FITC-labeled goat F(ab?)2anti-rabbit IgG
was obtained from Biosource and was used at 10 ?g/ml.
(iv) EBV-specific antibodies. Anti-BZLF1 MAb BZ.1 (56) was from Dako
(Denmark); anti-EA-D antibody R3 (41) was from ABI (Columbia, Md.); anti-
gp350/220 antibody 2L10 was obtained from G. Pearson; anti-LMP-1 MAb S12
was produced from the hybridoma (31). MAbs were used at 40 ?g/ml or accord-
ing to recommendations of the manufacturers and detected with FITC-labeled
goat F(ab?)2anti-mouse IgG (Biosource) at 10 ?g/ml. Affinity-purified rabbit
antibody specific for the carboxyl-terminal cytoplasmic domain of LMP-1 (2) was
generously provided by B. Sugden. A human anti-viral capsid antigen (VCA)
serum that was shown to recognize primarily late EBV antigens by immunoflu-
orescence assay was used at a 1:20 dilution.
Hybridization probes. Random-prime-labeled probes were prepared and
RNA blot hybridization was performed as described previously (8). To detect
LMP-1 mRNA, the 495-bp NcoI-NcoI fragment of exon C, positions 168263 to
168758 of EBV genomic DNA, was used. An LMP-2A cDNA was excised from
a pBluescript KS? clone by using EcoRI (45). For glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), a cDNA clone, pHcGAP (50), was obtained from the
ATCC. An EagI fragment of EBV DNA (66823 to 67633) was the U-exon probe.
pRA386 (54) was used to probe for EBER1.
RESULTS
Stable infection of 293 cells. To explore whether EBV could
stably infect cell lines of epithelial origin, we tested whether
EBV that had been genetically altered to carry a G418 resis-
tance gene could stably infect a given cell line and yield G418r
colonies. For most experiments, we used a recombinant EBV
designated P3-531, which was generated prior to each infection
by homologous recombination between P3HR1 virus and plas-
mid p531, resulting in replacement of the viral BHRF1 gene
with a CMVIE-neo gene (27). Stocks of P3-531 recombinants,
typically containing 40 to 200 infectious recombinants per ml,
were added to dishes containing subconfluent cultures of the
cell lines of epithelial origin, using up to 1 ml of P3-531 stock
per 106adherent cells. After 2 or 3 days, the culture medium
was replaced with medium containing G418, and the plates
were inspected periodically for emerging G418rclones. A small
number of G418rcolonies appeared reproducibly when the cell
2116FINGEROTH ET AL.J. VIROL.

Page 3

line 293 was tested. We failed to obtain any G418rcolonies
after attempting to infect another adenovirus-transformed ep-
ithelial cell line, RHEK, and three different cell lines derived
from human carcinomas, HeLa, D98, and HEp2.
The number of stably infected 293 clones that could be
obtained from a given virus stock was consistently lower than
would be expected from the titer of recombinant virus that
could be measured by infecting the Burkitt’s lymphoma-de-
rived cell lines Raji and BL30. As shown in Table 1, a stock of
P3-531 virus with a titer of 55 G418-resistance-conferring units
per ml, as measured by infecting BL30 cells, gave an effective
titer of only 1.3 for stable infection of 293 cells. The difference
was more dramatic for a G418-resistance-conferring virus de-
rived from the B95-8 strain of EBV. This recombinant virus,
B-652, has been recovered in the producer cell line, B95-8,
from which it and the parental B95-8 virus are released in
comparable amounts (27a). Two different stocks of B-652 virus
that were tested on the cell lines at different times could stably
infect the Burkitt’s lymphoma cell lines several hundred to a
few thousand times more efficiently than they could stably
infect 293 cells (Table 1).
EBV binds to CD21 on the surface of 293 cells. 293 cells
were examined for the presence of CD21 molecules at their
surface by using flow cytometry to measure the binding to
intact cells by rabbit antibodies specific for human CD21. As
shown in Fig. 1A, the CD21-specific serum produced a histo-
gram that was clearly shifted to a higher fluorescent intensity
than that obtained with normal rabbit serum. This result indi-
cated that most 293 cells contained CD21 molecules at their
surface. A similar result was obtained with the CD21-specific
MAb HB-5 compared to an irrelevant, isotype-matched con-
trol antibody (see Fig. 3). Proteins extracted from 293 cells
were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and immunoblotting using the
rabbit anti-CD21 serum (Fig. 1B). A protein with an apparent
size of 145 kDa was detected at the same positions as the
authentic CD21 glycoprotein extracted from the B-cell line
X50-7, although at a much lower quantity. CD21 was not
detected in extracts of an osteosarcoma-derived cell line, 143,
or an adenovirus-transformed human kidney epithelial cell
line, RHEK. We conclude that 293 cells contain authentic
CD21 at their surface.
The density of CD21 molecules at the surface of most 293
cells appears to be much lower than it is with two B-cell lines
that EBV readily infects, Raji and BL30 (data not shown). The
T-lymphoma cell line HPB-ALL, which expresses levels of
surface CD21 similar to those of the B-lymphoma lines Raji
and BL30, appeared to bind about the same amount of HB5
antibody as did 293 cells in the experiment of Fig. 1A. But 293
cells are larger than HPB-ALL cells and also bind more anti-
body nonspecifically (Fig. 1A), and so the surface density of
CD21 molecules must be significantly lower for 293 cells than
for HPB-ALL cells. The number of CD21 molecules per cell is
clearly much lower for 293 cells than it is for the EBV-immor-
talized cell line X50-7 (Fig. 1B and 3), where a high level of
CD21 is induced by expression of EBV type III latency gene
products.
If the uptake of EBV by a cell is to some extent dependent
on the surface density of CD21, then it is possible that EBV
infects 293 cells less efficiently than B cells because 293 cells
have a lower density of CD21 receptor molecules on their
surface. In this case, the stably infected clones of 293 cells
might have arisen from variants that expressed higher levels of
CD21. Two EBV-infected clones of 293 were examined for
CD21 content by immunoblot analysis and were found to have
perhaps twice as much CD21 as the uninfected 293 cell pop-
ulation (Fig. 1B). This could mean that there is some hetero-
geneity with respect to the level of CD21 in the uninfected
population and that cells having more CD21 than the popula-
FIG. 1. Detection of CD21 on 293 cells with a monospecific rabbit antiserum. (A) Detection of CD21 on the cell surface of 293 compared with HPB-ALL by flow
cytometry. Cells were stained either with rabbit anti-CD21 (block) or with normal rabbit serum (line). (B) Detection of CD21 protein in extracts of 293 cells by Western
analysis. Total soluble protein from Nonidet P-40 lysis of 500,000 cells was analyzed for the cell lines indicated. Two clones of 293 that are stably infected with the EBV
recombinant P3-531 are indicated as 293-5 and 293-3. At the left, positions are indicated (in kilodaltons) for standards.
TABLE 1. Abilities of 293 and two Burkitt’s lymphoma cell lines to
become stably infected with two different strains of EBV
Virus
Titer of G418 EBV (CFU/ml)aafter infection with:
BL30Raji293
P3-531
B95-652
B95-652
55ND
63,000
33,000
1.3
20
80
120,000
65,000
aMeasured as described in Materials and Methods. Two different stocks of
B95-652 were tested at different times. Repeated determinations were within
twofold of the average shown in each case. ND, not determined.
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tion average might have been selectively infected. It is also
conceivable that this slight increase in CD21 is a consequence
of low levels of EBV-encoded LMP-1 expressed in these cells
(see below), since LMP-1 has been shown to induce CD21
gene expression in B-cell lines (51).
To test whether binding of EBV to 293 cells could be de-
tected, purified EBV of strain B95-8 was labeled with FITC
and allowed to bind to 293 cells, which were then washed free
of unbound virus and examined by flow cytometry. A histo-
gram with a broad peak, perhaps with a shoulder at higher
fluorescent signal, was obtained (Fig. 2A). The histogram
shown is representative of results obtained from three inde-
pendent preparations of labeled virus, each analyzed at least
twice. The brighter fluorescence exhibited by about half of the
cells in the population was not observed if, before the addition
of fluoresceinated EBV, the 293 cells were treated with HB-5
followed by goat F(ab?)2specific to mouse IgG in order to
cross-link the CD21 molecules on the cell surface (Fig. 2B).
The cross-linking step had no effect on the binding of fluores-
ceinated EBV when the irrelevant MAb UPC10 was used
instead of HB-5 (Fig. 2C). This is evidence that EBV can bind
to a large fraction of a population of 293 cells and that this
binding is mediated by CD21.
CD21-specific antibodies block infection of 293 cells by EBV.
To examine whether the virus binding that is mediated by
CD21 is responsible for the cells becoming infected, we tested
whether antibodies specific to CD21 would block infection of
293 cells. To do this, 293 cells in six-well dishes were incubated
with the CD21-specific MAb OKB7, with an isotype-matched
control antibody, MOPC21, or with an anti-HLA class I mono-
clonal antibody, W6/32, for 30 min at room temperature
(24°C). The cells were then washed to remove antibody and
incubated with virus for 1 h before being washed and returned
to culture to monitor outgrowth of G418rclones. Alternatively,
after washing away the primary antibody, a secondary antibody
was added for 30 min in order to cross-link the receptor before
infection with EBV. As shown in Table 2, 10 to 23 infected
clones were generated from each infection when cells were
either not treated with antibody or treated with control anti-
body MOPC21, which does not bind to 293 cells, or W6/32,
which recognizes abundant class I HLA molecules at the sur-
face of 293 cells. However, when cells were treated with CD21-
specific OKB7, no infected clones emerged. OKB7 blocked
infection with or without the cross-linking step. Previously,
OKB7 had been shown to block EBV attachment to B cells
directly, without a need to cross-link with a secondary antibody
(36). A rabbit antiserum specific to CD21 also blocked infec-
tion with or without treatment with a cross-linking antibody,
while the preimmune serum from the same rabbit had little or
no effect. These results provide strong evidence that most or all
infections of 293 cells by EBV are mediated by CD21.
Examining 293 cells for the presence of proteins that form
complexes with CD21. On B-cell membranes, CD21 is reported
to reside in two distinct complexes, one with CD35 (CR1, the
receptor for complement component C3b) and one with CD19,
CD81 (TAPA-1), and Leu-13 (6, 48). It is possible that EBV
infects 293 cells less efficiently than B cells because CD21 is
present independently on 293 cell surfaces or in complexes
with different proteins at the surfaces of the different types of
cells. As shown in Fig. 3, we could not detect either CD35 or
CD19 on 293 cells. The 293 cells expressed a higher level of
CD81 (TAPA-1) than did X50-7 cells. Anti-Leu-13 gave a
slight shift in the histogram relative to the isotype-matched
FIG. 2. CD21-dependent binding of FITC-labeled EBV to 293 cells, as de-
termined by flow cytometry. (A) Binding of FITC-labeled EBV to 293 cells
without pretreatment. (B) Before incubating 293 cells with FITC-labeled EBV,
surface CD21 was cross-linked with anti-CD21 mouse MAb HB-5 followed by
goat F(ab?)2against mouse IgG. (C) 293 cells were treated identically except that
the irrelevant control MAb UPC10 was used for cross-linking.
TABLE 2. Blocking infection of 293 cells with antibodies
specific to CD21a
Antibody No. of G418rclones
1st2ndObtained Expectedb
Not applicable
(No virus)
None
MOPC21
W6/32
OKB7
MOPC21
W6/32
OKB7
Rabbit preimmune
Rabbit ?-CD21
Rabbit preimmune
Rabbit ?-CD21
0
16
23
15
019
?-cMouse IgG
?-Mouse IgG
?-Mouse IgG
10
18
0 14
14
0
12
14
?-Rabbit IgG
?-Rabbit IgG112
Total with ?-CD21159
aSee Materials and Methods for details.
bNumber or average number observed with the matched-antibody controls.
c?, anti.
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FIG. 3. Analysis of surface expression of CD21 and proteins reported to form membrane complexes with CD21 for 293 cells and X50-7 cells. Binding is indicated
(block) for mouse MAbs specific to CD21 (HB-5), to CD35/CR1 (543), to CD19 (B-C3), to CD81/TAPA-1 (5A6), and to Leu-13 (anti-Leu-13). Each is shown in
comparison to binding by an irrelevant antibody, UPC10 (line).
VOL. 73, 1999EBV INFECTION OF 293 CELLS2119