Fusion of epithelial cells by Epstein–Barr virus
proteins is triggered by binding of viral glycoproteins
gHgL to integrins ?v?6 or ?v?8
Liudmila S. Chesnokovaa,b,c, Stephen L. Nishimurad, and Lindsey M. Hutt-Fletchera,b,c,1
aDepartment of Microbiology and Immunology,bCenter for Molecular and Tumor Virology, andcFeist-Weiller Cancer Center, Louisiana State University
Health Sciences Center, Shreveport, LA 71130; anddDepartment of Pathology, University of California at San Francisco, San Francisco, CA 94143
Edited by Patricia G. Spear, Northwestern University, Chicago, IL, and approved October 12, 2009 (received for review July 7, 2009)
Epstein–Barr,virus (EBV) is a ubiquitous human herpesvirus that is
causally implicated in the development of lymphoid and epithelial
tumors. Entry of virus requires fusion of virus envelopes and cell
membranes. Fusion with B lymphocytes requires virus glycoprotein
interactions between glycoprotein 42 (gp42) and HLA class II. How-
ever, fusion with epithelial cells is impeded by gp42 and instead is
a 2-part complex of gHgL. We report here that gHgL binds with high
affinity to epithelial cells and that affinity of binding is increased by
integrin ?v, or by a peptide corresponding to 13 aa of gH which
include a KGDE motif. Fusion of cells expressing gB and gHgL can be
blocked by vitronectin or triggered by addition of soluble truncated
integrins ?v?6 and ?v?8. We conclude that the direct interaction
between EBV gHgL and integrins ?v?6 and ?v?8 can provide the
trigger for fusion of EBV with an epithelial cell.
tomatically in childhood, but, although primary infection in ado-
the major impact of the virus results from its role as a tumor
initiator or tumor progressor. EBV is associated with both lym-
these 2 cell types (1).
The proteins involved in EBV penetration of a B cell are more
clearly defined than those required for epithelial cells (reviewed in
glycoprotein gp350 and the complement receptor type 2 (CR2) or
CD21. Fusion, as for all herpesviruses, requires the core fusion
fusion machinery from a metastable to an active state requires an
interaction with HLA class II, which functions as a coreceptor or
to form a 3-part complex, gHgLgp42.
Attachment of EBV to epithelial cells can be mediated by gp350,
but on CR2-negative cells it also can be mediated by a 2-part
complex, gHgL. A soluble truncated form of the complex, gHtgL,
can bind specifically to epithelial cells but not to B cells (4), and a
loses the ability to bind to a CR2-negative epithelial cell (5, 6). In
addition, HLA class II, which is not constitutively expressed on
epithelial cells, is unavailable to trigger fusion. Instead, fusion with
an epithelial cell requires an unknown coreceptor, gB, and the
gHgL complex that lacks gp42. Virus carries both 3-part gHgLgp42
complexes and 2-part gHgL complexes to accommodate infection
of 2 cell types. Only 3-part complexes can mediate fusion with B
cells, but only 2-part gHgL complexes can mediate entry of epi-
thelial cells (7), and changes in the levels of gp42 by sequestration
and degradation in an HLA class II-positive B cell, but not in an
pstein–Barr virus (EBV) is carried by ?90% of the adult
population worldwide. Many individuals are infected asymp-
HLA class II-negative epithelial cell, switch virus tropism (8). The
to an epithelial cell that lacks CR2 (4) and also blocks the ability of
virus bound via gp350 to a CR2-positive cell to infect (7).
to a CR2-negative cell but also blocked entry of bound virus into
coreceptor needed to trigger epithelial cell fusion by EBV glyco-
proteins. We previously have speculated that this triggering might
in fact be the primary function of the gHgL receptor, because,
the ability of the virus to enter the cell when using the molecule,
rather than CR2, for attachment is not (4). We report here that 1
set of proteins that can function as gHgL receptors are ?v-
containing integrins. Downregulation of ?v expression reduced
binding and infection, as did a peptide corresponding to residues of
184–196 of the gH precursor, which include a putative integrin-
binding motif, KGDXXXL. Further, soluble forms of human
EBV gB and gHgL. We conclude that integrins are able to serve
both for attachment and fusion of EBV with epithelial cells,
although their most important role is likely to be in fusion.
Binding of gHtgL to Epithelial Cells Is Characteristic of Integrin
Binding. The CR2-negative AGS gastric carcinoma cell line and the
SVKCR2 keratinocyte cell line engineered to express CR2 have
been used extensively to study epithelial cell infection with cell-free
fusion; on SVKCR2 cells, gp350 can be used for attachment, and
gHgL is essential only for fusion (5). To characterize gHgL binding
as a result of production in insect cells, was isolated by affinity
chromatography on Lentil Lectin Sepharose. Biological activity of
gHtgL was monitored during isolation by flow cytometric analysis
of protein bound to cells on ice and stained with monoclonal
antibody CL59 to gH. Binding was saturable, fitted well to a
hyperbolic curve [see supporting information (SI) Fig. S1], and
could inhibit virus infection (Fig. S2). Isolated protein then was
labeled with125I and used for Scatchard analysis of binding to AGS
Author contributions: L.S.C. and L.M.H.-F. designed research; L.S.C. performed research;
S.L.N. contributed new reagents/analytic tools; L.S.C. and L.M.H.-F. analyzed data; and
L.M.H.-F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
December 1, 2009 ?
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no. 48 www.pnas.org?cgi?doi?10.1073?pnas.0907508106
by 1 order of magnitude were detected (Fig. 1 and Table 1); only
the high-affinity sites were found on SVKCR2 cells (Table 1).
The predicted 706-aa sequence of gH includes a KGD motif at
residues 188–191 that potentially could serve as a ligand for
integrins. Integrins can exist in an open or a closed conformation,
and the difference in affinity of the 2 conformations for ligand can
be as much as 9,000-fold (9). Because Mn2?locks integrins in an
measured. Addition of 200 ?M Mn2?had no effect on the number
of binding sites on AGS or SVKCR2 cells but increased their
affinity of binding by approximately 3 orders of magnitude (Fig. 1
and Table 1). To determine if the region of gH that included the
KGD motif might serve to bind gHtgL to the cells, binding was
added to facilitate determination of peptide concentration. The
cognate peptide, but not a scrambled form containing the same
residues, blocked gHtgL binding (Fig. 2). The cognate peptide also
reduced the ability of virus to infect SVKCR2 cells, although, as
might be expected for a peptide in competition with a multivalent
ligand, considerably more peptide was required to reduce infection
as much as 70% than was required to inhibit gHtgL binding.
Fibronectin and Vitronectin Reduce Binding and Infection, and Vitro-
nectin Inhibits Fusion. The natural ligands of integrins are matrix
proteins that also might be expected to compete with gHtgL for
binding. Preincubation of cells with fibronectin and vitronectin, but
not with BSA, competitively reduced gHtgL binding and infection
of SVKCR2 cells when gHgL was used only for fusion (Fig. 3 and
Fig. S3). Neither fibronectin nor vitronectin had any effect on
infection of B cells, which do not bind gHtgL (Fig. 3). Vitronectin
and gHgL and examined visually (Fig. 4). It also blocked fusion of
CHO-K1 cells transfected with plasmids encoding gB, gH, and gL.
gHtgL bound (fmol)
gHtgL Free (pmol)
gHtgL Bound (fmol)
gHtgL bound (fmol)
Bound/Free (10 )
gHtgL Free (pmol)
gHtgL Bound (fmol)
without affecting the number of binding sites. Scatchard analysis of125I-
Manganese increases the affinity of gHtgL binding to AGS cells
Table 1. Affinity and number of gHtgL binding sites on AGS and SVKCR2 cells
High-affinity binding sitesLow-affinity binding sites
(0.61 ? 0.90*) ? 10?9
(0.26 ? 0.06) ? 10?12
(0.91 ? 0.14) ? 10?9
(0.18 ? 0.02) ? 10?12
(286 ? 1.00) ? 103
(297 ? 36.00) ? 103
(51 ? 2.00) ? 103
(49 ? 1.00) ? 103
(6.0 ? 0.05) ? 10?9
(0.97 ? 0.22) ? 10?12
(8.41 ? 1.40) ? 106
(6.03 ? 0.01) ? 105
*Standard deviations were calculated from three separate experiments.
Peptide concentration (μM)
[125I]gHtgL binding (%)
Peptide concentration (μM)
Infected cells (% control)
Binding of125I-labeled gHtgL to cells preincubated with increasing concen-
inhibition of binding (r ? 0.91). Initial slopes of the 2 conditions are signifi-
cantly different (P ? 0.0001). (B) Infection of cells preincubated with the
indicated amounts of the cognate KGD peptide (solid line) or a scrambled
form of the same peptide (dotted line). The plot of the scrambled peptide
obeys a linear equation (r ? 0.73), and that for the cognate peptide obeys an
equation for competitive inhibition of binding (r ? 0.63). Initial slopes of the
2 conditions are significantly different (P ? 0.0003). Plots in both panels are
the averages of 3 independent experiments.
A peptide corresponding to residues 184–196 of gH blocks gHtgL
Chesnokova et al.PNAS ?
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vol. 106 ?
no. 48 ?
In this assay cells were cotransfected with a plasmid-expressing
luciferase under control of the T7 promoter and overlaid with
293T14 cells that express T7 polymerase. Fusion is measured in
the ability of CHO-K1 cells transfected with plasmids encoding gB,
gHgLgp42, and luciferase to fuse with Daudi29 B cells expressing
Silencing of ?v Integrins as Well as Soluble Forms of Human Integrins
That Interact with gHtgL Reduce gHtgL Binding and Infection. The
ability of vitronectin or fibronectin to affect binding and fusion
in gH is followed by a leucine residue at the plus-4 position, as is
expressed on epithelial cells (12) but not on B cells (13). SVKCR2
cells expressed 4 integrins known to bind vitronectin or fibronectin,
?5?1, ?v?5, ?v?6, ?v?8. The mean fluorescence intensities (MFI)
of antibody binding were 61.10, 27.56, 109.11, and 29.68, respec-
tively (Fig. S4). A fraction of the cells also expressed ?v?3 (MFI
29.47). AGS cells expressed ?5?1, ?v?5, and ?v?6 (MFI 44.82,
23.72, and 26.09, respectively) but not ?v?8 (MFI 4.02) or ?v?3
(MFI 3.78). Akata B cells, which do not bind gHtgL, also express
?5?1 (4), so it seemed probable that gHtgL was binding to an ?v
integrin. To test this possibility, SVKCR2 cells were transfected
with cognate but not control siRNA (MFI reduced from 198.93 to
20.45; Fig. 5A), and binding of gHtgL also was reduced (MFI
reduced from 46.69 to 28.14). Importantly, reduction of expression
of ?v also reduced infection by EBV expressing GFP, dropping the
MFI of infected cells from 1115.47 to 163.64 (Fig. 5B). Although
vitronectin blocked gHtgL binding well, gHtgL failed to bind to
Binding of125I-labeled gHtgL to AGS cells in medium alone (none) or in the
presence of 40 ?g/mL of BSA (BSA), fibronectin (FN), or vitronectin (VNR). (B)
Infection of SVKCR2 cells in the presence of medium alone or 40 ?g/mL BSA,
fibronectin, or vitronectin. (C) Infection of EBV-negative Akata B cells in the
presence of medium alone or 40 ?g/mL BSA, fibronectin, or vitronectin. Error
bars indicate SD of 3 infections and duplicate binding assays.
Fibronectin and vitronectin block gHtgL binding and infection. (A)
was added immediately after transfection of AGS cells with gB and gHgL (AG-
S:AGS), after transfection of CHO-K1 cells with gB, gHgL, and a luciferase con-
struct and subsequently overlaid with 293T14 cells (CHO:293T14), or after trans-
fection of CHO-K1 cells with gB, gHgL, gp42, and a luciferase construct,
subsequently overlaid with Daudi29 cells (CHO:Daudi29) (black bars). Fusion is
expressed as a percentage of that occurring in the absence of vitronectin (white
bars). Error bars indicate SD of 3 experiments; the SD of AGS :AGS fusion in the
presence of vitronectin is too small to visualize.
% of Max
% of Max
were nucleoporated with siRNA to ?v (dashed line) or with control siRNA (solid
line), and expression of ?v (Left) or binding of gHtgL (Right) was measured by
flow cytometry. The isotype controls are shown as dotted lines. (B) SVKCR2 cells
expression of ?v (Left) or infection (Right) was measured by flow cytometry.
Isotype controls or uninfected cells are shown as dotted lines. Experiments are
representative of 3 different nucleoporations/transfections.
www.pnas.org?cgi?doi?10.1073?pnas.0907508106Chesnokova et al.
CHO cells expressing human ?v?3 (Fig. S5). Culture media of
CHO cells engineered to secrete a soluble form of human ?v?6
blocked gHtgL binding to SVKCR2 cells, reducing the MFI from
Preincubation of virus with soluble forms of integrins ?v?6 and
?v?8 made in engineered 293 cells reduced infection, but a soluble
form of ?v?3 made in the same cell type did not (Fig. 6B). An
interaction between gHtgL and the soluble integrins ?v?6 and
?v?8, but not between gHtgL and ?v?3, also could be observed by
reciprocal immunoprecipitation (Fig. 7).
Soluble Human Integrins That Interact with gHtgL Trigger Fusion of
Cells That Express gB and gHgL but Lack Human Integrins. CHO-K1
cells, which do not bind gHtgL, are used for cell-based fusion
assays because they do not fuse with each other when transfected
with gB and gHgL. To determine if the interactions of integrins
?v?6 and ?v?8 with gHgL were able to trigger epithelial cell
fusion, these integrins were added to CHO-K1 cells 4 h after
transfection of cells with plasmids expressing gB and gHgL; 20 h
later cells were inspected visually for evidence of fusion. Soluble
even in the presence of 1 mM Mn2?(Fig. S6). Preincubation of
?v?6 and ?v?8 with cognate-blocking antibodies reduced the
extent of fusion (Fig. 8).
Many viruses, including herpesviruses, use integrins to facilitate
entry into mammalian cells (14). The interactions can serve to
attach virus to cells (15), to stimulate endocytosis (16), and to
induce signaling pathways that are important to infection (17, 18).
Interaction of an EBV envelope protein, BMRF2, with integrins
(19). We show here that the interaction with integrins of another,
essential envelope protein, gH, is directly responsible for triggering
fusion of epithelial cells by EBV gB and gHgL.
only herpesviruses that carry potential integrin-binding motifs in
gH. The gH homolog of the alphaherpesvirus, herpes simplex virus
(HSV), includes an RGD sequence at residues 176–178 of the
predicted 803-aa protein. Low-affinity binding assays confirmed
that in this context the motif can bind to ?v?3 integrins (22), but
mutation of the sequence had no effect on virus penetration (23).
Instead, in all cell types currently examined, HSV fusion more
closely resembles fusion of EBV with a B cell, in that it requires not
only the core fusion machinery (gB and gHgL) but also gD.
Although gD binds to several different cell proteins or sulfated
l l e c d
e t c e f n I
) l o r t n
none αvβ3 αvβ6 αvβ8
% of Max
integrins. (A) Flow cytometric analysis of gHtgL binding to SVKCR2 cells in the
presence of fresh tissue culture media (light solid line), concentrated spent
soluble human ?v?6 (heavy solid line). Cells were preincubated with super-
natants before addition of gHtgL. The isotype control is shown as a dotted
line. (B) Infection of SVKCR2 cells with virus preincubated with soluble ?v?3,
?v?6, or ?v?8. Error bars indicated the SD of triplicate infections.
Integrins or gHtgL were immunoprecipitated (IP) individually or together as
of each integrin (A) or with antibody to gH (B) and were Western blotted (WB)
with antibody to ?v (Upper) or gH (Lower).
Fused cells (%)
overlaid with integrins, and 20 h later were fixed and stained with monoclonal
nuclei (% fusion) after overlay with integrins (black bars) or integrins preincu-
bated with cognate blocking antibody (gray bars). Error bars indicate SD of 5
experiments without antibody and 2 experiments with antibody.
Soluble ?v?6 and ?v?8 trigger fusion of CHO-K1 cells transfected with
Chesnokova et al.PNAS ?
December 1, 2009 ?
vol. 106 ?
no. 48 ?
analogous to that of gp42 (3).
Rather than broaden the spectrum of interactions of which
gp42 is capable, as seems to have happened for gD, EBV has
adopted a fundamentally different and more direct approach to
activate fusion with an epithelial cell. The question that thus
arises—how 2 different cell proteins, HLA class II and an
integrin, result in activation of what is thought to be ultimately
the same fusion machinery—is similar in some ways to that
raised by the ability of so many different structures to have the
same downstream effect when they interact with gD. However,
gH, unlike HSV gD, is thought to play an intrinsic role in fusion
by inducing hemi-fusion (24), and thus there presumably are
even more functional constraints on the ways in which gH can
interact with a trigger than there are for gD. The binding of gp42
to gH before fusion with a B cell (25, 26) suggests that it is gH
that is used to transmit, to the entire fusion machinery, the
conformational changes that are likely to occur following HLA
class II binding to gp42 (27); this notion is consistent with the
ability of a direct interaction between gH and an integrin to
activate a fully functional fusion machinery. Mutational analysis
has distinguished sequences in gH that are differentially impor-
tant for fusion with a B cell and an epithelial cell (25, 28),
suggesting that some events downstream of the integrin–gH
interaction may be distinct from those initiated by a gp42-HLA
class II complex. However, the assumption that the ultimate
effects on gH are the same seems reasonable.
addressed here. It remains possible that integrins in addition to
?v?6 and ?v?8 can bind and function as fusion inducers. Attempts
to identify these integrins by use of function-blocking antibodies
have been only partially illuminating. Antibodies to ?1 or ?5?1, 2
other integrins (in addition to the non-interactive ?v?3) that are
Antibody to ?v?5, ?v?6, ?v?8, and ?v alone reduced binding by
as well. However, the possibility that the effect was mediated by
interference of this antibody with ?v cannot be ruled out com-
pletely, because we know nothing about the nature of the interface
of gH and any integrin. A combination of antibodies decreased
binding beyond that mediated by antibody to ?v alone, but the
combination could not eliminate binding completely, possibly be-
as the affinity of many antibodies, or perhaps because there are
additional cell surface molecules with which gHgL can interact.
S7B). We do know, however, in the case of integrin binding, that
both ? and ? integrin subunits must be human, because gHtgL did
not bind to CHO cells expressing hamster ?v and human ?6 (Fig.
S8). Determination of the full range of integrins that can be used
to trigger fusion will require generation of reagents that currently
are unavailable. The extent to which infection of human cell lines
integrins together with the entry-competent attachment receptor
CR2 also will require the generation of novel reagents. CR2 is
expressed on relevant epithelial cells in vivo (29), and the extent of
its dominant role in facilitating efficient infection is still unclear.
It was somewhat surprising that a soluble integrin alone was able
to stimulate fusion, but we assume that cell–cell interactions were
themselves sufficient to put gB and gHgL close enough to an
adjacent cell that, once activated, they could mediate fusion. This
assumption suggests that the binding of gH to an integrin results in
a significant conformational change, a possibility that will be
explored further. The fact that it was possible to block infection by
to be productive.
?v?8, most particularly ?v?6, is intriguing. Expression of ?v?8 in
epithelial cells is limited primarily to basal cells in normal airway
normal epithelial tissues in vivo, and its expression is up-regulated
during tissue remodeling, including that accompanying wound-
healing, inflammation, and carcinogenesis (31, 32). Functionally, in
addition to roles in mediating interactions with the extracellular
matrix, ?v?6 and ?v?8 bind to TGF-?1 latency-associated peptide
and in doing so cause local activation of endogenous TGF-?1 (11,
which in turn enhances matrix deposition and fibrosis. This effect
may be of interest, given the association of EBV with idiopathic
pulmonary fibrosis (34–36). Critically, however, TGF-?1 also in-
duces EBV lytic reactivation in B cells (37–39). There thus are 2
reasons why up-regulation of ?v?6 on dysplastic cells might en-
hance the chances of virus transfer from any proximal B cell
carrying EBV. If, as has been suggested, a role of EBV in devel-
opment of tumors such as nasopharyngeal carcinoma is to induce
progression in a cell that already has undergone molecular alter-
ations (40, 41), the consequences may not be benign.
Materials and Methods
Cells. AGS, SVKCR2, EBV-negative Akata, Akata-GFP, and Sf9 insect cells have
in RPMI MEDIUM 1640 with 10% heat-inactivated serum. CHO-K1 cells VNRC3
(CHO cells expressing human ?v?3) (43), F4B6 (CHO cells expressing human ?6),
express T7 RNA polymerase) (45), and 293-B8 AVAP (11), 293-B6 AVAP, and
293-B3 AVAP (cells that, respectively, secrete truncated ?v?8, ?v?6, and ?v?3
conjugated to alkaline phosphatase) (46) were grown in DMEM supplemented
with 10% heat-inactivated FBS and 1% nonessential amino acids.
Antibodies and Immunofluorescence. Monoclonal antibodies used include CL59
to gH (5); E1D1 to gHgL (47); CL55 to gB (28); 37E1 to ?v?8 (11); L230 (44) to
National Institute of Dental and Craniofacial Research); BV3 to integrin ?v?3;
Sc-8066 to alkaline phosphatase (Santa Cruz); andAV1 to integrin ?v, R6G9 to
integrin ?6, 10D5 to integrin ?v?6, P1F6 to integrin ?v?5, and JBS5 to integrin
?5?1 (all from Chemicon). CL59, E1D1, CL55, 37E1, and L230 were purified by
affinity chromatography on protein A Agarose (RepliGen). Rabbit anti-peptide
were prepared for flow cytometry (4) or indirect immunofluorescence as de-
scribed in ref. 28. See SI Methods for more details.
Virus and Virus Infection. EBV-GFP and baculovirus expressing gHtgL (49) were
made as described in ref. 4. Infection of epithelial cells or B cells was done as
described in ref. 4, and the effects of peptides and cell matrix proteins were
examined by preincubation of cells with reagents for 1 h at 4 °C. See SI Methods
for more details.
Isolation of Soluble gHtgL. SolublegHtgLwasobtainedfromculturemediumof
Sf9 cells infected with recombinant baculovirus as described in ref. 4, concen-
trated by ultrafiltration, and dialyzed to remove sugars. gHtgL was isolated on
side. See SI Methods for more details.
Isolation of Soluble Truncated Integrins. Serum-freespentculturemediumfrom
CHO- or 293-cell derivatives was concentrated by ultrafiltration. Fractions con-
chromatography on Superdex 200(GE Healthcare). The concentrations of these
to ?v and secondary antibody coupled to HRP, detected with ECL reagent (GE
Healthcare) and fluorography and scanned into ImageQuant (GE Healthcare).
gHtgL Binding and Scatchard Analysis. Binding was done as described in ref. 4.
Binding also was measured after gHtgL was radiolabeled with125I. Specific
bound to cells following preincubation with a saturating amount of unlabeled
gHtgL. Nonspecific binding never exceeded 7% of total binding. The range of
www.pnas.org?cgi?doi?10.1073?pnas.0907508106 Chesnokova et al.
gHtgL concentrations used for Scatchard analysis was chosen to cover 0.1/10 KD Download full-text
after calculation of an approximate KDfrom saturation curves obtained by flow
out normalizing. Numeric data were obtained using EnzFitter 1 (Biosoft). See SI
Methods for more details.
Silencing. SVKCR2 cells, as indicated, either were nucleofected with 15 nmol of
siRNAs to human ?v or negative control siRNA obtained from Ambion using an
Amaxa nucleoporator (Lonza), the V kit, and program T20 or were transfected
with 2 ?M siRNA using Dharmafect 1 reagent (Dharmacon) according to the
manufacturer’s instructions. gHtgL binding, integrin expression, and infection
were measured by flow cytometry.
Cell Fusion Assays. Cell fusion assays were done as described by transfection of
45). Triggering of CHO-K1 cell fusion without overlaying cells was achieved by
adding isolated integrins 4 h after transfection followed by visual examination
antibody for 1 h before being added to cells. See SI Methods for more details.
Immunoprecipitation and Western Blotting. Proteins were immunoprecipitated,
electrophoresed, and transferred to PVDF membranes as described in ref. 5,
except that proteins were run under nonreducing conditions. The membranes
were reacted with primary antibody and secondary antibody coupled to HRP
(Amersham) for detection by ECL (GE Healthcare) and fluorography.
ACKNOWLEDGMENTS. We thank Susan Turk for excellent technical assistance,
expressing human ?6 and soluble human ?v?6 and for helpful discussion, and
the gift of monoclonal antibody 13. This work was supported by National Insti-
tute for Dental and Craniofacial Research Grants DE016669 (to L.M.H.-F) and
HL63993 and NS04415 (to S.L.N.).
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