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INTRODUCTION |
The expression of viral proteins that counter immune responses of
the host is well documented. Viral factors have been identified that
can potentially inhibit or modify the antiviral effects of antibodies,
complement proteins, cytokines, and cytotoxic T cells (1).
Characterization of viral proteins that interact with specific
components of the immune system is likely to provide insights into
immune mechanisms involved in host-virus interactions and into the
molecular basis of viral persistence in the presence of a functional
immune system. Herpesviruses, in particular, have evolved
multiple mechanisms for interfering with humoral as well as
cell-mediated immune responses (reviewed in Refs. 2 and 3). The present
studies focus upon the herpes simplex virus type I-encoded Fc receptor
(FcR),1 a protein complex
that has been suggested to interfere with antibody-mediated viral
clearance (4). HSV-1 virions, as well as cells infected with HSV-1,
bind to immunoglobulins of the IgG subclass via the Fc region (5). The
glycoprotein gE of HSV-1 was identified as the IgG-binding polypeptide
of HSV-1 (6, 7). It was subsequently shown that gE associates with a
second viral glycoprotein, gI (8, 9), and that cells transfected with
genes encoding both gE and gI have enhanced IgG binding activity
compared with cells transfected with gE alone (10-12). Both gE and gI
are type I transmembrane proteins, with an N-terminal extracellular
portion, a single transmembrane domain, and a C-terminal cytoplasmic
domain. Homologous glycoproteins encoded by other
-herpesviruses,
including pseudorabies virus (PRV) (13) and varicella zoster virus
(14), have been shown to possess species-specific FcR activity.
HSV-1-infected cells acquire low levels of FcR activity immediately
upon exposure to virus (in the absence of viral gene expression), presumably by the transfer of virion gE-gI to the cell surface during
viral entry (7). The HSV FcR may thus be particularly significant for
protection of virally infected cells from early immune destruction (2).
Recent in vivo studies demonstrated that passively
transferred anti-HSV IgG greatly reduced viral titers and disease
severity in mice infected with a mutant HSV-1 that lacked FcR activity.
By contrast, anti-HSV IgG was ineffective in reducing viral titers and
disease severity in mice infected with wild type virus with intact FcR
activity (15). These observations indicate that the HSV-1 FcR activity
facilitates evasion of antibody-mediated viral clearance in
vivo.
Several means of evading antibody-mediated immune responses could arise
from the Fc binding function of gE-gI (16-18). Binding of nonimmune
IgG by gE-gI present on HSV-1 virions can inhibit virus neutralization
by anti-viral antibodies (19). Engagement of the Fc portion of anti-HSV
antibodies can protect virally infected cells from
antibody-dependent cell-mediated cytotoxicity (ADCC) (20)
as well as complement-mediated lysis (21). Inhibition of ADCC has been
suggested to occur by a phenomenon called antibody bipolar bridging
(21), a mechanism whereby antibodies bound via their Fab ends to HSV-1
glycoproteins on surface membranes of infected cells would
simultaneously interact with the viral Fc receptors of the same
infected cell. By engaging the Fc domain, the HSV-1 FcR could interfere
with recognition by Fc
Rs on immune effector cells. Antibody bipolar
bridging has also been suggested to facilitate antiviral
antibody-induced patching, capping, and extrusion of viral
glycoproteins from the surface of cells infected with PRV (13).
Antibody-induced shedding of viral glycoproteins may represent a
strategy for rendering virally infected cells refractory to antiviral
antibodies and for inhibiting the presentation of viral antigens via
class II major histocompatibility complex molecules.
A second function attributed to gE-gI is that of facilitating
cell-to-cell spread of virus. Recent studies suggest that gE and gI are
required for transneuronal transport of PRV from the retina to the
visual centers of rats (22), for cell-to-cell spread of PRV, and for
full virulence of PRV (23). Furthermore, studies with mutant HSV-1
virions indicate that gE and gI of HSV-1 facilitate cell-to-cell spread
of virus in vivo and viral spread across junctions of
cultured cells (24-26). It has been proposed that the cell-to-cell
spread-promoting functions of gE-gI are unrelated to the Fc
binding activity and that the HSV-1-encoded gE-gI glycoproteins and the
analogous proteins of other
-herpesviruses may interact with
other ligands, enabling viral transport across cells (24, 25). Such
ligands remain to be identified.
To better understand the mechanisms by which IgG binding by gE-gI
facilitates immune evasion, we initiated a molecular characterization of IgG binding by gE-gI. We expressed soluble forms of gE and gI and
showed that the glycoproteins assemble into a stable heterodimer. The
soluble receptor heterodimer binds to human IgG (hIgG) with relatively
high affinity and can be purified to homogeneity using an hIgG-based
affinity matrix. We determined a 1:1 binding stoichiometry for the
gE-gI·IgG complex, and also determined that a histidine residue at
the CH2-CH3 domain interface is a critical
determinant of IgG binding specificity. The implications of the gE-gI
binding site on IgG and the gE-gI·IgG complex stoichiometry are discussed.
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EXPERIMENTAL PROCEDURES |
Construction and Expression of Soluble gE, gI, and
gE-gI--
Molecular cloning manipulations were performed by standard
protocols (27). PCR was used to insert a 5' XhoI site, a 3'
NotI site, and a stop codon after the codon corresponding to
amino acid 399 of the gE gene and amino acid 246 of the gI gene (the HindIII fragment containing the gE gene and the
BamHI fragment containing the gI gene of HSV strain KOS was
kindly provided by H. Ghiasi, Cedar Sinai Medical Center). Our
numbering scheme starts with the first residue of the mature protein,
which is designated residue 1, and all other residues are numbered
sequentially (see "N-Terminal Sequencing and Mass Spectrometric
Analysis of Purified gE-gI"). The gE PCR product was cloned into
pCRII (Invitrogen), and the gI PCR product was cloned into
pBSIISK+ (Stratagene). Both sequences were verified. The
modified gE and gI genes were excised using XhoI and
NotI enzymes and individually subcloned into the unique
XhoI and NotI sites of separate PBJ5-GS expression vectors (28). PBJ5-GS carries the glutamine synthetase gene
as a selectable marker and as a means of gene amplification in the
presence of the drug methionine sulfoximine, a system developed by
Celltech (29). Expression vectors carrying gE, gI, or both gE and gI
were transfected into CHO cells using a Lipofectin procedure (Life
Technologies, Inc.). Cells resistant to 100 µM methionine sulfoximine were selected according to the protocol established by
Celltech, modification of which has been previously described (28).
Transfected CHO cells were maintained in glutamine-free
-minimal
essential medium (Irvine Scientific) supplemented with 5% dialyzed
fetal bovine serum (Life Technologies), 100 µM methionine sulfoximine (Sigma), penicillin (100 units/ml), and streptomycin (100 µg/ml). Cells secreting gE, gI, or both gE and gI were identified by
immunoprecipitation of supernatants of cells metabolically labeled with
[35S]methionine and [35S]cysteine (see
below) by using either an antibody against gE (1108 (Goodwin Institute)
or Fd172 (30) (kindly provided by Subbu Chatterjee) or an antibody
against gI (Fd69 (31), kindly provided by Subbu Chatterjee) Clones were
considered positive if immunoprecipitation yielded a protein of
approximately 56 kDa corresponding to gE or a protein of approximately
43.5 kDa corresponding to gI. The identity of each protein was verified
using N-terminal sequencing (see below).
35S Metabolic Labeling--
gE-, gI-, and
gE-gI-transfected CHO cell lines derived from colonies were expanded
into 12-well trays, grown to confluence, and incubated for 5 h in
1.0 ml of methionine- and cysteine-free medium (Life Technologies) plus
1% dialyzed fetal bovine serum including 5 µCi of a
[35S]methionine and [35S]cysteine (ICN)
mixture. Supernatants were clarified by a 5-min spin in a
microcentrifuge, and either anti-gE or anti-gI antibodies were added.
Immunoprecipitations were carried out by standard methods (32) with
protein G-bearing Sepharose beads (Amersham Pharmacia Biotech). Samples
were boiled in SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
running buffer and loaded onto 15% polyacrylamide gels, which were
fixed, dried, and exposed to a PhosphorImager screen (Molecular
Dynamics, Inc., Sunnyvale, CA). The image was then developed with a
Molecular Dynamics 425E PhosphorImager scanner.
Co-Expression of Full-length gE and gI--
PCR was used to
insert a 5' XhoI site and a 3' NotI site into the
genes encoding gE and gI. The PCR products were sequenced and
subsequently individually subcloned into the unique XhoI and NotI sites of separate PBJ5-GS expression vectors. The two
constructs were co-transfected into CHO cells, and cells resistant to
100 µM methionine sulfoximine were selected. Cells
expressing both gE and gI were sorted using fluorescence activated cell
sorting (FACS) analysis with FITC-labeled hIgG. Individual clones from the sort were amplified and subsequently shown to express both gE and
gI by FACS analysis with 1108 or Fd69 as the primary antibodies and a
goat anti-mouse IgG as the secondary antibody. Sorting and analysis
were performed on a Coulter Epics Elite flow cytometer.
Purification of Soluble gE-gI Heterodimers--
gE-gI-secreting
CHO cell lines were grown to confluence in 50 10-cm plates and
introduced into a hollow bioreactor device (Cell Pharm I; Unisyn
Fibertec, San Diego, CA) in serum-free medium, and supernatants were
collected daily. Soluble gE-gI heterodimers were purified from
supernatants on either a human Fc or hIgG affinity column. The human Fc
column was prepared by coupling 20 mg of human Fc (Jackson
ImmunoResearch Laboratories, Inc.) to cyanogen bromide-treated
Sepharose 4B (Amersham Pharmacia Biotech) at approximately 10 mg of
protein/ml of resin according to the protocol of the manufacturer. The
hIgG column was prepared similarly using 70 mg of hIgG (Sigma).
Supernatants were passed over the affinity column, which was then
washed with 50 column volumes of a solution consisting of 50 mM Tris (pH 7.4), 0.1% NaN3, and 1 mM EDTA. Bound gE-gI was eluted from the column with 50 mM diethylamine (pH 11.5) into tubes containing 1.0 M Tris (pH 7.4). gE-gI heterodimers were further purified
using a Superdex 200 HR 10/30 fast protein liquid chromatography (FPLC)
filtration column. Approximately 10 mg of gE-gI heterodimers were
recovered per liter of transfected cell supernatants.
N-terminal Sequencing and Mass Spectrometric Analysis of Purified
gE-gI--
N-terminal sequencing was performed on 2.5 µg of
purified, soluble gE-gI in a phosphate buffer dried onto a
polyvinylidene difluoride membrane and inserted into an Applied
Biosystems model 476A sequencer reaction cartridge. Two sequences were
isolated from the gE-gI sample: the sequence GTPKTSWRR, corresponding
to the first 9 amino acids of mature gE (33), and the sequence LVVRGPTVS, corresponding to the first 9 amino acids of mature gI (33).
The molecular masses of gE and that of gI were determined by
matrix-assisted, laser desorption, time-of-flight mass spectrometry with a PerSeptive biosystems (Farmington, MA) ELITE mass spectrometer.
CD Analyses--
An AVIV 62A DS spectropolarimeter equipped with
a thermoelectric cell holder was used for CD measurements. Wavelength
scans and thermal denaturation curves were obtained from samples
containing 10 µM protein in 5 mM phosphate at
pH 7 by using a 0.1-mm path length cell for wavelength scans and a 1-mm
path length cell for thermal denaturation measurements. The
heat-induced unfolding of gE-gI was monitored by recording the CD
signal at 223 nm, while the sample temperature was raised from 25 to
80 °C at a rate of approximately 0.7 °C/min. The transition
midpoint (Tm) for unfolding was determined by
taking the maximum of a plot of d
/dT versus T (where
is ellipticity) after averaging the data with a
moving window of 5 points.
Gel Filtration Analyses of gE-gI·hIgG
Stoichiometry--
Protein concentrations were determined
spectrophotometrically at 280 nm using the following extinction
coefficients: gE-gI, 88816 M
1
cm
1; hIgG, 202,500 M
1
cm
1. The extinction coefficient for the gE-gI heterodimer
was calculated from the amino acid sequences as described (34), and the
extinction coefficient for hIgG is known (32).
A280 measurements for a fixed amount of each
protein were then compared in 6 M guanidine HCl and aqueous
solutions, and the extinction coefficients were adjusted as necessary.
For determining the gE-gI·hIgG stoichiometry, various molar ratios
from 1:3 (300 pmol of gE-gI:900 pmol of hIgG) to 3:1 (900 pmol of
gE-gI:300 pmol of hIgG) of gE-gI and hIgG were incubated for 30 min at
room temperature in 20 mM Tris, pH 7.4, 150 mM
NaCl, 0.05% NaN3 in a total volume of 100 µl. Samples were injected onto a Superose 6B FPLC column (Amersham Pharmacia Biotech) and eluted with the same buffer at 0.5 ml/min. The composition of each fraction was analyzed by SDS-PAGE (data not shown).
Equilibrium Analytical Ultracentrifugation--
Sedimentation
equilibrium was performed with a Beckman Optima XL-A analytical
ultracentrifuge, using data analysis software provided by the
manufacturer. Experiments were performed using 0.6 mg/ml gE-gI at both
4 and 20 °C at a rotor speed of 10,000 rpm, with equilibrium times
of at least 36 h. Molecular masses were determined by nonlinear
least square fit of the equilibrium gradient, absorbance
versus radius (Fig. 3), using the model of single ideal
species, and a partial specific volume, 0.69, calculated from the amino
acid composition and the carbohydrate content (35).
Equilibrium Column Chromatography--
The equilibrium column
chromatography method of Hummel and Dreyer (36) was used to observe the
interaction between gE-gI and hIgG. A Superdex 200 PC 3.2/30 gel
filtration column of 2.4 ml was connected to an Amersham Pharmacia
Biotech µ Precision pump system. Absorbance of the eluant was
monitored at 280 nm with an Amersham Pharmacia Biotech µ Peak
monitor. The column was equilibrated with five different concentrations
of purified hIgG (Sigma): 250 nM, 500 nM, 1 µM, 2.5 µM, and 5 µM each in
20 mM Tris, pH 7.4, 150 mM NaCl. At each
concentration, four 20-µl injections in the appropriate column
equilibration buffer (including the relevant concentration of hIgG)
were performed. These four injections included gE-gI at a concentration
equal to that of the IgG in the column buffer plus no additional hIgG
or hIgG at a concentration equal to 1, 2, or 3 times that of the IgG
concentration contained in the column buffer. Binding experiments were
done at 20 °C with a flow rate of 100 µl/min.
Biosensor Studies--
Biosensor studies were performed on a
Biacore 2000 instrument. Purified gE-gI was diluted in 10 mM acetate buffer, pH 4.1, for amine-based coupling to a
Biacore chip. Immobilization was accomplished by initial activation of
the sensor chip with 0.2 M
N-ethyl-N'-(dimethylaminopropyl)-carbodiimide and
0.05 M N-hydroxysuccinimide. The
N-hydroxysuccinimide-ester was then reacted with gE-gI using the manual injection mode to allow for better control of immobilization levels. Typically, an immobilization level of 200-300 response units
was used for kinetic analyses described in Table I. The remaining
unreacted ester groups were inactivated by 1 M ethanolamine (pH 8.5). Different concentrations of the chimeric IgG molecules were
injected over the immobilized gE-gI surface, as well as a control
protein surface (murine IgG). A citrate buffer, pH 3.5, was used for
regeneration of the surface between sequential injections. Sensorgrams
obtained for IgG binding to the control surface were subtracted from
those obtained for IgG binding to the gE-gI surface. The BIAevaluation
version 3.0 software package was used for kinetic analysis. Kinetic
constants were derived by simultaneous fitting to the association and
dissociation phases of the subtracted sensorgrams and global fitting to
all curves in a working set (Fig. 5). A working set consisted of
injections of four or five different concentrations of a hIgG construct
over a surface containing immobilized gE-gI. S.D. values are
reported from experiments performed in duplicate or triplicate on
different sensor chips (Table I). In all cases, a 1:1 binding model was
used for curve fitting.
The expression of chimeric hIgG molecules was described previously (37,
38). These molecules are composed of a murine anti-dansyl
VH domain fused to the constant domains (CH1
through CH3) of hIgG4. An expression vector containing
cDNA encoding the hybrid chain was co-transfected into a
non-Ig-producing mouse myeloma line along with an expression vector
containing cDNA encoding a chimeric K light chain (composed of a
murine anti-dansyl VK region fused to the human
CK region). Site-directed mutations were introduced into
the chimeric heavy chain gene to make the hIgG4H435R mutant and the
hIgG3R435H mutant. The hIgG3-hIgG4 chimeras were generated by exon
shuffling as described previously (38).
Determination of KD Values by Cell Binding
Assays--
Chimeric hIgG4 was iodinated to a specific activity of
16.1 µCi/µg using the chloramine-T method. CHO cell lines
expressing full-length gE and gI were grown to confluence in tissue
culture plates. Cells were detached by incubation with
phosphate-buffered saline (pH 7.5), 5 mM EDTA for 20-30
min and collected in binding buffer at pH 7.0 (Hanks' balanced salt
solution, 10 mM HEPES, 0.25% bovine serum albumin). The
cells were pelleted, washed once with binding buffer (pH 7.0), and
resuspended in binding buffer (pH 7.0). Cells (1 × 106) were mixed in duplicate or triplicate assays with
labeled hIgG4, different concentrations of unlabeled hIgG4, and binding
buffer (pH 7.0) to a total volume of 0.5 ml. The samples were incubated for 2 h at room temperature. After completion of the incubations, cells were pelleted for 5 min at 14,000 rpm in an Eppendorf
microcentrifuge, the supernatants were aspirated, and 1.0 ml of cold
binding buffer was added. After removal of the supernatants by
aspiration, the tubes were placed in vials, and the levels of
radioactivity were determined using a Beckman Gamma 5500 counter.
Nonspecific binding was determined by a similar treatment of wild type
CHO cells. The binding data were analyzed using Scatchard plots. Assays
were performed in triplicate, and the average of the two most similar readings was used to compute the concentration of bound IgG.
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RESULTS |
Co-Expression of Truncated gE and gI Results in Assembly of a
Stable Heterodimer--
We constructed soluble versions of both gE and
gI by truncating each of the genes prior to their predicted
transmembrane regions (following the codons for amino acid 399 of
mature gE and amino acid 246 of mature gI). The modified genes were
co-transfected into CHO cells. Transfected cells were screened by
immunoprecipitating supernatants from metabolically labeled cells with
antibodies against either gE or gI (Fig.
1A). SDS-PAGE analysis of
immunoprecipitated protein from gE-gI positive clones revealed two
bands with apparent molecular masses of 56 and 43.5 kDa using either
the anti-gE or anti-gI antibody. The calculated molecular mass of
truncated gE is 42 kDa, and that of gI is 26 kDa; however, both
proteins are glycosylated (two potential N-linked
glycosylation sites in the sequence of gE and three potential sites in
the sequence of gI) and would be expected to migrate with a higher
apparent molecular mass.

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Fig. 1.
Soluble gE and gI assemble into a stable
complex. Cells producing gE, gI, or both gE and gI or
nontransfected CHO cells were labeled with
[35S]methionine/cysteine, and cell supernatants were
analyzed. A, SDS-PAGE (10%) analysis of protein isolated
from supernatants of 35S-labeled cells producing gE, gI, or
gE-gI using antibodies against either gE (lanes 1 and 2) or gI (lanes 3 and
4). B, SDS-PAGE (10%) analysis of gE, gI, and
gE-gI binding to either Sepharose-immobilized rat IgG (lanes
1-4) or hIgG (lanes 5-8).
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HSV-1-infected cells have previously been shown to encode proteins that
bind hIgG but not rodent IgG (39). To investigate the binding
characteristics of soluble gE, gI, and the gE-gI heterodimer, metabolically labeled supernatants from gE-, gI-, or gE-gI-secreting cells were incubated with Sepharose-immobilized hIgG or rat IgG. SDS-PAGE analysis revealed that while none of the proteins bind rat
IgG, the gE-gI complex efficiently bound to the hIgG matrix. gE alone
bound only weakly to the human IgG matrix, while gI alone showed no
specific interaction (Fig. 1B).
A purification scheme based upon the FcR activity of gE-gI was used to
isolate soluble gE-gI heterodimers for biochemical studies.
Supernatants from cells expressing gE-gI were passed over an hIgG
affinity column, eluted at high pH, and then further fractionated by
size exclusion chromatography using a Superose 6B gel filtration
column. A single homogenous peak corresponding to a gE-gI complex was
obtained, demonstrating that any free gE or gI present in the
supernatants does not efficiently associate with the hIgG matrix. By
contrast, gE is not purified when supernatants from cells expressing
only gE are passed over the hIgG column.
Soluble gE-gI migrates on the gel filtration column slower than
predicted by the molecular mass of a 1:1 heterodimer (100 kDa). Indeed,
the retention time for gE-gI is greater than that for IgG (165 kDa)
(Fig. 2C). The increased
retention might arise because gE-gI is not a 1:1 heterodimer or because
of anomalous migration of a 1:1 heterodimer with an elongated or
otherwise nonspherical shape. In order to determine the stoichiometry
of soluble gE-gI, we analyzed the protein by N-terminal sequencing and
equilibrium analytical ultracentrifugation. N-terminal sequencing of
purified gE-gI confirmed the presence of the correctly processed forms
of both proteins in approximately stoichiometric amounts (data not
shown). The molecular mass of soluble gE-gI determined by equilibrium
analytical ultracentrifugation is 83.4 kDa (Fig. 3), in close agreement with the predicted
molecular mass of a 1:1 gE-gI heterodimer calculated using molecular
masses of each monomer determined by mass spectrometry (gE, 48.4 kDa;
gI, 33.5 kDa). To investigate the stability of soluble gE-gI, we used a circular dichroism-based thermal unfolding assay, from which we determined that the heterodimer denatures cooperatively with a Tm of 66 °C (data not shown).

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Fig. 2.
Stoichiometry determination of the
gE-gI·IgG complex using conventional gel filtration. gE-gI and
IgG were incubated for 30 min at pH 7.4 at the indicated molar ratios
and then passed over a size exclusion column to separate the
gE-gI·IgG complex from uncomplexed proteins. At a 1:1 molar ratio of
gE-gI to IgG, all of the protein chromatographs as a single complex.
When the input ratio of gE-gI to IgG is greater than 1:1, there is
excess gE-gI (A), whereas when the input ratio of IgG to
gE-gI is greater than 1:1, there is excess IgG (B) (verified
by SDS-PAGE analysis; data not shown). C, gE-gI and IgG each
elute as a single peak and can be distinguished from one another on the
basis of their retention times.
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Fig. 3.
Sedimentation equilibrium analysis of
gE-gI. gE-gI at 0.6 mg/ml was centrifuged at 10,000 rpm until
equilibrium was reached (36 h). The gradient formed can be best fit to
a single species with a mass of 83.4 kDa. The errors of the fit, shown
in the residuals plot, are small and random.
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Taken together, these results indicate that gE-gI is a stable
heterodimer with 1:1 stoichiometry and that the heterodimer, but
neither free gE nor free gI, binds to monomeric hIgG with high
affinity. Thus, the observed interaction of free gE with IgG reported
here (Fig. 1B, lane 5) as well as
previously (10) must be low affinity or specific for aggregated IgG
(11).
The Stoichiometry of the gE-gI·IgG Complex Is 1:1--
The
stoichiometry of the gE-gI·hIgG complex was determined to be 1:1
using a non-equilibrium-based gel filtration assay and confirmed using
an equilibrium column chromatography method (36). As shown in Fig. 2,
gE-gI, IgG, and the gE-gI·hIgG complex each elute as single peaks
from a Superose 6B column and can be distinguished from one another on
the basis of their retention times. To determine the stoichiometry of
the gE-gI·IgG complex, various molar ratios of gE-gI to IgG were
pre-equilibrated and then passed over the Superose 6B column. When
gE-gI and IgG were present at equimolar ratios, a single peak
corresponding to the gE-gI·IgG complex eluted from the column (Fig.
2, A and B). SDS-PAGE analysis of the eluted material revealed that both gE-gI and IgG were present in the peak
(data not shown), indicating that gE-gI and IgG form a stable complex
under these conditions. When the input ratio of gE-gI to IgG was
greater than 1:1, a peak corresponding to excess gE-gI was observed in
addition to the gE-gI·IgG complex peak, whereas a peak corresponding
to excess IgG was observed in addition to the complex peak when the
input ratio was less than 1:1 (Fig. 2, A and
B).
To verify the 1:1 stoichiometry of the gE-gI·hIgG complex, we also
used an equilibrium-based method. In this method, a gel filtration
column was equilibrated with buffer containing a uniform concentration
of hIgG (equilibration buffer). gE-gI and hIgG mixtures in
equilibration buffer were injected over the gel filtration column. Four
injections were made, containing gE-gI at a concentration equal to that
of hIgG in the equilibration buffer and either no additional hIgG or 1, 2, or 3 mol eq of hIgG. In all cases, all of the injected gE-gI binds
to hIgG, migrating as the gE-gI·IgG complex. When the amount of
additional hIgG injected is less than or greater than the amount
required for formation of the gE-gI·IgG complex, a trough (in the
case of too little hIgG) or a peak (in the case of excess hIgG) should
be observed at the position where free hIgG migrates. When the amount
of additional hIgG injected is equal to that required for formation of
the gE-gI·IgG complex, a flat base line should be observed at the
position where hIgG migrates. Over the concentration range from 250 nM (Fig. 4A) to 5 µM (Fig. 4B), injections of additional hIgG in
an amount equivalent to that of gE-gI in the sample result in a flat
base line at the hIgG migration position. These results verify that the
stoichiometry of the gE-gI·hIgG complex is 1:1 over a protein
concentration range of 250 nM to 5 µM.

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Fig. 4.
Stoichiometry determination of the
gE-gI·IgG complex using equilibrium gel filtration. A Superdex
200 column was equilibrated with 20 mM Tris, 150 mM NaCl, pH 7.4 containing either 250 nM hIgG
(A) or 5 µM hIgG (B). A,
250 nM gE-gI was injected in equilibration buffer (20 mM Tris, 150 mM NaCl, pH 7.4, 250 nM hIgG) along with the indicated additional concentrations
of hIgG. B, 5 µM gE-gI was injected in
equilibration buffer (20 mM Tris, 150 mM NaCl,
pH 7.4, 5 µM hIgG) along with the indicated additional
concentrations of hIgG.
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Residue 435 at the CH2-CH3 Domain Interface
Is Critical for gE-gI·IgG Binding--
Previous IgG binding studies
with HSV-1-infected cells indicated that hIgG1, hIgG2, and hIgG4 bind
to the HSV-1 FcR, while many hIgG3 allotypes do not bind (39-41). This
subtype binding preference resembles the binding preferences for IgG
binding by Staphylococcus aureus protein A (protein A) and
certain classes of rheumatoid factors (RF; antibodies that bind to the
Fc portion of Ig) (38, 42-45). We used biosensor-based assays to
quantitate the affinity between gE-gI and hIgG subtypes and to
characterize the molecular basis of the observed binding specificities.
Purified soluble gE-gI was immobilized on the surface of a Biacore
biosensor chip using an amine-based coupling chemistry, as described in the Biacore Methods manual. We analyzed the binding of chimeric murine-hIgG molecules composed of the variable domains of a murine anti-dansyl immunoglobulin fused to the constant domains of hIgG1, hIgG2, hIgG3, or hIgG4 (37, 38). The chimeric hIgG subtypes were
analyzed for binding to immobilized gE-gI at low coupling densities of
gE-gI (100-300 response units), conditions under which mass
transport-limited binding is not significant (46). The derived binding
constants are summarized in Table I.
Chimeric hIgG1, hIgG2, and hIgG4 bind to immobilized gE-gI with
equilibrium dissociation constant (KD) values of
200-400 nM. Of the different hIgG subtypes, hIgG4 has the
highest affinity for gE-gI, while hIgG3 does not show detectable
binding (>5 response units) at concentrations up to 3.0 µM (Table I and Fig. 5,
A and B).
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Table I
Binding of hIgG constructs to gE-gI immobilized on a Biacore chip
Kinetic constants were derived from sensorgram data using simultaneous
fitting to the association and dissociation curves and global fitting
to all curves in a working set. Kinetic analysis was performed using
the BIAevaluation version 3.0 package. The equilibrium constants,
KD, were determined from the ratios of the kinetic
constants. For hIgG3, hIgG4H435R, and 3-4-3-3, signals of less than 4 RU were observed at protein concentrations of 3.0 µM.
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Fig. 5.
Biosensor analysis of the binding of hIgG3,
hIgG4, and the corresponding residue 435 mutants. Soluble gE-gI
was immobilized on the surface of a Biacore chip using a primary
amine-based coupling protocol. The injected samples were 188 nM to 1.5 µM hIgG3 (A), 46-366
nM hIgG4 (B), 70.8-566 nM
hIgG3R435H (C), or 250 nM to 2 µM
hIgG4H435R (D). For each set of binding experiments,
sensorgrams are overlaid with the calculated response using a 1:1
binding model. One representative set of injections from experiments
performed in duplicate or triplicate is shown for each
interaction.
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For an independent verification of the biosensor-derived affinities,
full-length gE and gI were expressed in CHO cells (Fig. 6), and the binding affinity for hIgG4
was derived using iodinated hIgG4. Scatchard analysis of the binding
data yields a KD value of 40.4 ± 13 nM, compared with 199 ± 35 nM in the
biosensor-based analysis (Fig. 7). The
5-fold lower affinity determined using the biosensor assay could
reflect that covalent immobilization of gE-gI results in reduced
affinity for IgG or that the membrane-bound form of gE-gI has a higher
affinity for IgG than the soluble version. Although biosensor assays
may underestimate the true binding affinity of gE-gI for IgG, they
allow quantitative comparison of the relative binding affinities of
different hIgG mutants for gE-gI.

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Fig. 6.
Expression of full-length gE and gI in CHO
cells. CHO cells transfected with genes encoding gE and gI were
assayed for surface expression of both proteins using flow cytometry.
Untransfected (dotted lines) or transfected
(solid lines) cells were stained with
FITC-labeled human IgG (A), the mouse anti-gE antibody 1108 followed by FITC-labeled goat anti-mouse antibody (B), or
the mouse anti-gI antibody fd69 followed by FITC-labeled goat
anti-mouse antibody (C). In B and C,
the dashed lines represent staining of
transfected cells with FITC-labeled goat anti-mouse antibody
alone.
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Fig. 7.
Cell binding assay for determination of the
binding affinity of membrane-bound gE-gI for IgG. 1 × 106 CHO cells expressing membrane-bound gE-gI were
incubated with different concentrations of 125I-labeled
chimeric hIgG4. Binding data are presented as a Scatchard plot. Each
point represents the average of two duplicate measurements. Three
independent experiments yielded an average binding constant of 40 ± 13 nM.
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Position 435 of hIgG sequences contains a polymorphism that
distinguishes many hIgG3 allotypes from hIgG1, hIgG2, and hIgG4. In
many hIgG3, residue 435 is an arginine; a histidine is found in all
other subclasses. Histidine 435 is a contact residue for protein A
binding to IgG Fc (47) and is also important for the binding of some
rheumatoid factors to IgG (38, 48, 49). The inability of some RF to
recognize hIgG3 can be reversed if the G3m(st) allotype is used.
Recognition of this hIgG3 correlates with the presence of histidine at
position 435, while nonbinding hIgG3 have an arginine at position 435 (38, 48). Histidine 435 is located at the interface between the
CH2 and the CH3 domains of IgG (Fig.
8). The CH2-CH3
domain interface of IgG has previously been implicated as the binding
site for HSV-1 FcR, based upon inhibition studies with a proteolytic
fragment of protein A (50). Alteration of residue 435 could therefore
account for the observed differences in gE-gI binding to the hIgG
isotypes. Alternatively, since hIgG3 has an extended hinge compared
with other hIgG isotypes, hinge-proximal structural differences might
account for the observed subtype-specific binding preferences. To
investigate these possibilities, we used biosensor assays to examine
the binding of gE-gI to mutant hIgG3 and hIgG4 proteins and switch
variants in which the constant domains of different subclasses were
exchanged by exon shuffling (37, 38).

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Fig. 8.
The location of histidine 435 on the
structure of human Fc. A ribbon diagram of
the CH2 and CH3 domains of hIgG are shown
(47). The side chain of histidine 435 is shown on the carbon-
backbone. The figure was prepared using Molscript (75)
and rendered using Raster 3D (76).
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To investigate the effect of residue 435 upon gE-gI·IgG affinity, the
binding of gE-gI to hIgG3 and hIgG4 mutants was examined. The hIgG3
mutant, hIgG3R435H, contains a histidine at residue 435 in place of an
arginine in the wild type protein, while the hIgG4 mutant, hIgG4H435R,
contains an arginine at residue 435 in place of the histidine in the
wild type protein (38). The single residue change of arginine to
histidine at residue 435 of hIgG3 is sufficient to restore binding from
undetectable in the case of the wild type protein to an affinity of
947 ± 533 nM in the case of the single site mutant
(Fig. 5, A and C, and Table I). The reciprocal
change in hIgG4, histidine to arginine at position 435 (hIgG4H435R),
results in no binding at concentrations up to 3 µM, as
compared with a binding affinity of 199 ± 35 nM for
wild type hIgG4 (Fig. 5, B and D, and Table
I).
To probe for differences in affinity due to hinge-proximal structural
effects, the binding of gE-gI to switch variants of IgG was examined.
As described previously, switch variants have been generated by
exchanging the constant domains of different subclasses by exon
shuffling (38). The switch variants used in these studies were 3-3-4-4 (CH1 and hinge domains of hIgG3, CH2, and
CH3 domains of hIgG4), 4-3-4-4 (CH1,
CH2, and CH3 domains of hIgG4, hinge of hIgG3),
and 3-4-3-3 (CH1, CH2, and the CH3 domains of hIgG3, hinge of hIgG4). Biosensor assays indicate that the
switch variants 3-3-4-4 and 4-3-4-4 bind gE-gI with an affinity comparable with that of wild type hIgG4 (Table I). By contrast, the
switch variant 3-4-3-3 does not bind at concentrations up to 3.0 µM (Table I). These results demonstrate that histidine 435 at the CH2-CH3 domain interface of IgG is
critical for gE-gI binding and that the presence of the extended hinge
in the chimeric hIgG3 does not significantly hinder binding.
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DISCUSSION |
We have initiated a molecular characterization of IgG binding by
the herpesvirus gE-gI protein. gE and gI are known to associate in
HSV-1-infected cells and upon co-expression in heterologous systems (8,
9). Here we show that gE and gI assemble into a stable complex when
expressed as soluble proteins. We also show that the soluble gE-gI
complex can be purified to homogeneity based upon its Fc receptor
function, using IgG affinity chromatography. Gel filtration and
analytical ultracentrifugation experiments establish that soluble gE-gI
is a 1:1 heterodimer, consistent with observations for gE-gI complexes
derived from other
-herpesviruses (e.g. varicella zoster
virus (51)). These results demonstrate that the transmembrane and
cytoplasmic domains of gE and gI are not required for gE-gI heterodimer
assembly and that the extracellular domains are sufficient for the
assembly of gE and gI into a stable heterodimer.
Whereas neither gE nor gI alone efficiently bind monomeric hIgG, the
gE-gI heterodimer binds hIgG with relatively high affinity. Biosensor-based studies using immobilized gE-gI show that soluble gE-gI
binds to hIgG1, hIgG2, and hIgG4 with affinities in the range of
200-400 nM. Results from binding assays using CHO cells expressing membrane-bound gE-gI are in close agreement with the binding
constant of 50 nM reported for the interaction of rabbit IgG with HSV-1-infected cells (52). In addition, the observed binding
specificities for the gE-gI interaction with different hIgG subclasses
and rodent IgG parallels the binding specificities reported for IgG
interaction with HSV-1-infected cells (39, 53). Thus, our results
confirm that the FcR activity induced by HSV-1 infection of cells
corresponds to IgG binding by cell surface gE-gI heterodimers. The
relatively high affinity interaction between gE-gI and the hIgG
subtypes 1, 2, and 4 indicates that nonimmune monomeric hIgG can coat
HSV-1 virions at the high concentrations of hIgG present in serum
(60-70 µM), thereby inhibiting virus neutralization by
antiviral antibodies.
Antibody bipolar bridging by gE-gI on HSV-1-infected cells has been
implicated in inhibition of ADCC mediated by mammalian Fc
Rs (20).
Human peripheral blood mononuclear cells have been shown to mediate
lower levels of ADCC activity against target cells infected with wild
type HSV-1 compared with cells infected with a gE-negative HSV-1. These
differences were attributed to the engagement of the Fc regions of cell
surface-associated antibodies by cognate gE-gI rather than by the
Fc
R present on opposing immune effector cells. Among mammalian
receptors, Fc
RI (KD ~0.5 × 10
9 M) is a high affinity receptor, and
Fc
RII and Fc
RIII (KD <1 × 10
6 M) are low affinity receptors (reviewed
in Refs. 54 and 55). Thus, based upon affinity considerations alone,
the formation of a gE-gI·IgG complex is likely to inhibit ADCC
mediated by Fc
RII and Fc
RIII. In addition, the observed 1:1
stoichiometry for IgG interaction with gE-gI, Fc
RI, and Fc
RIII,
as well as the fact that only one of two available binding sites on IgG
is a high affinity site in the FcRn-IgG complex (reviewed in Refs. 54 and 55), suggests a marked asymmetry in the Fc regions of
receptor-bound IgGs, such that only one of the binding sites is in an
optimal conformation for binding to many receptors. The observed 1:1
stoichiometry of the gE-gI·IgG complex at micromolar concentrations
of the proteins indicates that the asymmetry of the gE-gI·IgG complex
may prevent high affinity interaction with a second gE-gI molecule.
Similar mechanisms could also account for a reduced reactivity of the gE-gI·IgG complex with other Fc
binding proteins, irrespective of
the binding site location.
The 1:1 stoichiometry of the gE-gI·IgG complex could have
implications for signaling mediated by IgG binding to cell surface gE-gI. Specifically, binding of monomeric IgG would not be expected to
induce dimerization of gE-gI heterodimers. However, aggregated IgG
(such as IgG in immune complexes) or anti-gE and anti-gI antibodies could result in gE-gI multimerization. In addition, IgG involved in
antibody bipolar bridging (21) could result in the oligomerization of
gE-gI with other viral glycoproteins. A conserved YXX(L/V) motif is observed in the cytoplasmic domains of gE from HSV-1, HSV-2,
and PRV (33, 56, 57). In mammalian receptors, the YXX(V/L)
motif is responsible for various signaling events such as the
internalization of endocytic receptors from the plasma membrane,
protein targeting to various cellular compartments (58), mediation of
immune cell activation (59), and inhibition of cellular immune
responses (60). The importance of the YXXL motif in
mammalian immune responses raises the question of whether the gE-encoded YXXL motif is functional in signal transduction
mediated by Fc binding. The FcR activity of gE-gI has been suggested to initiate signaling events that facilitate capping and extrusion of PRV
glycoproteins, induced by a polyclonal mixture of porcine anti-PRV
antibodies (13). Whether anti-HSV antibodies can mediate glycoprotein
capping and extrusion in HSV-infected cells remains an important
question to be addressed. If antibody-induced capping and extrusion of
viral glycoproteins occurs in HSV-1-infected cells, the importance of
Fc binding by gE-gI for the occurrence of the process can be rigorously
investigated with the current knowledge of gE-gI binding specificities
for different IgG and the interaction stoichiometry. These studies will
allow a better understanding of the mechanisms by which the FcR
activity of
-herpesviruses could modify the protective effects of
antiviral antibodies.
Antibody bipolar bridging has been implicated in inhibition of ADCC by
HSV (20) as well as in anti-PRV antibody-mediated glycoprotein capping
and extrusion (13); but is antibody bipolar bridging sterically
probable? Can an IgG molecule simultaneously use its Fab and the Fc
regions in interactions with antigens and Fc receptors? Although direct
evidence for the occurrence of antibody bipolar bridging is lacking,
fluorescence energy transfer studies indicate that the IgG molecule is
highly flexible (61), suggesting that simultaneous interactions of the
Fab and Fc domains as postulated in antibody bipolar bridging are feasible.
Using mutant forms of hIgG, we show that histidine 435 at the interface
between the CH2 and CH3 domains of IgG is
critical for the binding interaction. Other proteins known to interact at the CH2-CH3 domain interface include protein
A (47), protein G (62), the neonatal Fc receptor (63), and RF (44).
Crystal structures have been reported for Fc complexes with protein A (47), protein G (64), neonatal Fc receptor (65), and a Fab fragment
derived from a human IgM RF antibody (RF-AN) (49, 66). From comparisons
of the binding characteristics observed for the gE-gI·IgG complex and
IgG complexes with protein A, protein G, neonatal Fc receptor, and RF,
it appears that the gE-gI·IgG complex most closely resembles IgG
complexes with certain rheumatoid factors. The similarities include a
lack of binding of several hIgG3 allotypes, the species specificity
(binding to human and rabbit IgG, but lack of binding of rodent IgG
(39, 53, 66)), and the importance of histidine 435 in the binding
interaction. That the IgG binding specificity of gE-gI closely
resembles that of some RF is significant in understanding the origin of
RF, since it has been suggested that some RF arise as anti-idiotypic
antibodies against antibodies to bacterial or viral Fc
-binding
proteins, in a process known as idiotypic networking (44, 67, 68).
Anti-idiotypic antibodies recognize the idiotypic determinants
expressed in the V region of a particular antibody or the V regions of
a group of related antibodies. It has been proposed that anti-idiotypic
antibodies are expressed in order to regulate the expression of
antibodies that dominate the response to a particular antigen (69).
Suppression of B cells expressing these dominant antibodies would allow
for the proliferation of other antibodies using alternative V region
sequences and ultimately to the diversification of the antibody
response (70). While the expression of anti-idiotypic antibodies would
normally decline with the decreased expression of the antibodies to
which they are responding, anti-idiotypic antibodies that cross-react
with something so ubiquitous as self-IgG have the potential to be
continually propagated. This model of idiotypic suppression provides a
possible explanation for the production of RF as a result of HSV-1
infection. Expression of gE-gI on the virion and on the surface of
HSV-1-infected cells would lead to production of anti-gE-gI antibodies
and subsequently to the production of anti-anti-gE-gI antibodies that
have the potential to be RF if the epitope recognized by the anti-gE-gI antibody is the region on gE-gI that interacts with IgG-Fc. In addition, persistence of HSV-I infection may lead to continual production of RF.
The similarities we observe between the gE-gI·IgG complex and IgG
complexes with certain classes of RF support the hypothesis that some
RF might be anti-idiotypic antibodies against antibodies to gE-gI and
provide the basis to more closely examine the linkage between
herpesviral infections and pathogenic RF production. Further support
comes from studies by Tsuchiya et al. (71), which show that
some RF share idiotypic determinants with gE-gI, suggesting that these
RF may be anti-idiotypic antibodies against antibodies to gE-gI.
However, whether the observed similarities in binding characteristics
of gE-gI·IgG and the RF-IgG complexes will correspond to similarities
in the interactions at the atomic level remains to be determined from
crystallographic comparisons of gE-gI·IgG with RF·IgG complexes
that show the closest resemblance in binding characteristics.
Recent studies suggest that antibodies are highly protective against
herpes infections in human neonates (72). Based upon the binding
studies reported here and previous studies with HSV-1-infected cells
(39, 40, 53), gE-gI can mitigate the effects of antiviral antibodies of
the IgG1, IgG2, and IgG4 subtypes, whereas IgG3 allotypes might confer
the greatest protection to a host due to the inability of many IgG3
allotypes to bind gE-gI. HSV-specific antibodies of the IgG1, IgG3, and
IgG4 subclasses have been detected in genital herpes infections (73).
Because human neonatal Fc receptor (the receptor responsible for
trans-placental IgG transfer (74)) binds the four human IgG isotypes
with similar affinities,2
IgG3 is likely to be transferred to the fetus with equal efficacy compared with the other isotypes that are generated, and it may constitute the isotype that confers the greatest protection against neonatal herpes. However, it is possible that anti-HSV hIgG3 antibodies are not produced or are not effective in certain HSV infections, and
therefore, a virus expressing an IgG3-binding FcR would not experience
a selective advantage. This may explain the lack of hIgG3 binding to
gE-gI and the evolution of the viral FcR with specificity for other
hIgG subclasses.