Previous Article | Next Article 
J Virol, July 1998, p. 5937-5947, Vol. 72, No. 7
School of Dental
Medicine,1
Center for Oral Health
Research,2 and
School of Veterinary
Medicine,3 University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Received 5 March 1998/Accepted 14 April 1998
Previously, we showed that truncated soluble forms of herpes
simplex virus (HSV) glycoprotein D (gDt) bound directly to a truncated
soluble form of the herpesvirus entry mediator (HveAt, formerly HVEMt),
a cellular receptor for HSV. The purpose of the present study was to
determine the affinity of gDt for HveAt by surface plasmon resonance
and to compare and contrast the kinetics of an expanded panel of gDt
variants in binding to HveAt in an effort to better understand the
mechanism of receptor binding and virus entry. Both HveAt and gDt are
dimers in solution and interact with a 2:1 stoichiometry. With HveAt,
gD1(306t) (from the KOS strain of HSV-1) had a dissociation constant
(KD) of 3.2 × 10 Herpes simplex virus (HSV)
glycoprotein D (gD) is a viral envelope glycoprotein that has been
studied extensively by immunological, biochemical, and genetic
approaches (5, 9, 23, 31, 33, 50). It is one of the
essential glycoproteins for virus entry, and much evidence indicates
that it functions by interacting with a cellular receptor (3, 16,
17, 21, 25, 50).
Recently, expression cloning was used to isolate and identify a HeLa
cell gene product that allows for entry of many HSV strains when it is
expressed in normally nonpermissive Chinese hamster ovary (CHO) cells
(25). This gene product, the herpesvirus entry mediator
(HveA, formerly HVEM), is a type I integral membrane protein. Two other
cellular receptors for HSV entry that are not related structurally to
HVEM/HveA have been identified (12, 19, 49). Therefore, the
nomenclature for naming HSV receptors has been unified (49).
Truncated HveA (HveAt) contains a motif of four cysteine-rich
pseudorepeat sequences and is a member of the tumor necrosis factor
receptor superfamily (1, 24, 25, 37). HveA can be
considered a cellular receptor for HSV, and gD can be considered
the receptor binding protein because (i) HveA allows virus entry
into normally nonpermissive cells (25), (ii) antibody
against HveA blocks virus entry (25), (iii) soluble HveAt
binds specifically to gD in purified virus and blocks entry (32,
50), and (iv) soluble truncated gD (gDt) interacts specifically with HveAt in vitro (50). Furthermore, the native
conformation of gD is critical for this interaction whereas the
N-linked oligosaccharides (N-CHO) are not (50), as was
predicted by previous structure-function studies (reviewed in reference
7).
Our approach to defining the relationship between the structure and
function of gD has been to generate and examine a panel of mutations
(5, 22, 23, 27, 28, 33, 38-40). The genes for a number of
these variant proteins have been cloned into a baculovirus expression
system to produce truncated, soluble forms of the proteins that can be
purified in the absence of detergent and easily studied (31, 33,
36, 50).
We have compared the abilities of the different gDt variant proteins to
bind to soluble HveAt by enzyme-linked immunosorbent assay (ELISA)
(25, 50). In particular, a gD-1 variant with a deletion in
functional region IV, gD1( To characterize the binding of gDt to HveAt in real time, optical
biosensor technology was used. Biomolecular interaction analysis was
used to quantitate binding affinities (dissociation constants
[KDs]) and obtain values for kinetic on
(kon) and off (koff)
rates. The biosensor uses surface plasmon resonance (SPR) and changes
in refractive index to measure interactions between biomolecules at the
surface of a sensor chip (18, 29). Binding data are obtained
in real time and in the absence of labeled protein. SPR has been used
to study the binding of influenza virus hemagglutinin to sialic acid
receptors (42), soluble intercellular cell adhesion molecule
to human rhinovirus (4), and HSV gB to glycosaminoglycans (52).
The purpose of the present study was to determine the affinity of gDt
for HveAt and to compare and contrast the kinetics of an expanded panel
of gDt variants in binding to HveAt in an effort to better understand
the mechanism of receptor binding and virus entry. Furthermore, we
sought to explain why a series of insertion variants in functional
regions I to IV of gD failed to complement the infectivity of a gD null
virus in trans. Specifically, we looked at differences in
affinities for HveAt among the gDt variants and determined if
differences in KDs were due to changes in
kon, the rate of association of the complex,
koff, an indication of the stability of the
complex, or a combination of both.
Cells and viruses.
Sf9 (Spodoptera frugiperda)
cells (GIBCO BRL), used for producing recombinant baculoviruses and
recombinant glycoproteins, were propagated in Sf900II medium
(GIBCO BRL) (53).
Construction of recombinant baculoviruses expressing the cysteine
variants.
DNA fragments were generated by PCR with plasmids pWW201
(Cys 1,5), pDL143 (Cys 2,6), and pWW220 (Cys 3,4) (23) as
templates and with the primers used to construct bac-gD1 (306t)
(36). Each PCR product encoded a gD1 variant with one
disulfide bond completely removed, with a truncation at amino acid 306 before the transmembrane region, and with a six-histidine C-terminal tail. The PCR products constructed with pWW201 (Cys 1,5), pDL143 (Cys
2,6), and pWW220 (Cys 3,4) were each ligated into the transfer vector
pVTBac (33, 36, 53) to produce plasmids pCP262, pCP261, and
pCP263, respectively, and recombined into baculovirus (33, 36,
53). The viruses were designated bac-gD1 (cys1,5),
bac-gD1 (cys2,6), and bac-gD1 (cys3,4), and the proteins were
designated gD1 (cys1,5), gD1 (cys2,6), and gD1 (cys3,4).
gD1 (cys1,5) has Ser replacing Cys at positions 66 and 189, gD1 (cys2,6) has Ser replacing Cys at positions 106 and 202, and
gD1 (cys3,4) has Ser replacing Cys at positions 118 and 127.
Production and purification of gDt.
Detailed protocols exist
elsewhere for purification of gDt (36, 53). Briefly, Sf9
insect cells (GIBCO BRL) were grown in 3 liters of suspension cultures,
infected with recombinant viruses at a multiplicity of infection of 4, and cultured at 27°C in a Celligen Bioreactor (New Brunswick
Scientific) for 48 h. Cells were removed by centrifugation, and
the clarified medium was concentrated to 1 liter with a Pellicon
Cassette Filter (Millipore) with a 10,000-molecular-weight cutoff. The
concentrated supernatant was then exchanged with 6 liters of
phosphate-buffered saline (PBS) with the same tangential-flow system.
The exchanged medium was passed over a DL6 immunosorbent column and
eluted as previously described (33, 34, 36). Proteins were
concentrated, dialyzed against PBS, and stored at Production and purification of HveAt.
Mature HveA is
245 amino acids long (25). A soluble form of HveA truncated
at amino acid 200, just prior to the transmembrane region (HveAt), was
produced from recombinant baculovirus-infected insect cells and
purified by nickel-affinity chromatography as previously described
(50).
Molecular weight analysis. (i) Gel filtration.
Dextran blue
was used to determine the void volume of a Superdex 75 column and a
Superdex 200 column (HR 10/30; Pharmacia), and the columns were
calibrated with High and Low Molecular Weight Gel Filtration
Calibration Kits (Pharmacia Biotech) with PBS as the running buffer.
Two hundred microliters of a 1-mg/ml solution of gD1 (306t),
gD2 (306t), and gD1 (QAAt) was eluted from the Superdex 75 column, and the retention time was measured. gD1 ( (ii) Mass spectrometry.
To determine the mass of
gD1 (306t), a 30-µg/ml sample was analyzed by matrix-assisted
laser-desorption ionization and time of flight mass spectrometry
(Fisons Instruments VG TofSpec) (15) as previously described
(34, 50).
(iii) STEM.
A sample of gD1 (306t) in PBS was diluted to
5 µg/ml with 50 mM ammonium acetate and analyzed by scanning
transmission electron microscopy (STEM). STEM was performed at the
Brookhaven National Laboratory (46). The sample was
processed and analyzed as previously described (45).
ELISA.
ELISA was used to monitor the binding of gDt to HveAt
as previously described (50).
Binding of gDt to HveAt as detected with an optical
biosensor.
SPR experiments were carried out on a BIACORE X or
BIACORE 2000 optical biosensor (Biacore AB) at 25°C. The running
buffer for the experiments was PBS (0.1 M sodium phosphate, 0.15 M
NaCl; Pierce) containing 0.005% Tween 20 (PBS-T; pH 7.0).
Approximately 2,000 response units (RU) of purified HveAt was coupled
to flow cell 1 (Fc1) of a CM5 sensor chip via primary amines according to the manufacturer's specifications. Fc2 was activated and blocked without the addition of protein. To characterize the binding of gDt
variants to HveAt, the flow path was set to include both flow cells,
the flow rate was 50 µl/min, and the data collection rate was set to
high. Protein samples were serially diluted in PBS-T. Binding of each
gDt sample was allowed to occur for 2 min, with the wash delay set for
an additional 2 min to allow for a smooth dissociation curve. The chip
surface was regenerated by injecting brief pulses of 0.2 M sodium
carbonate, pH 9.5, until the response signal returned to baseline. SPR
data were analyzed with BIAevaluation, version 3.0, software, which
employs global fitting. Sensorgrams were corrected for nonspecific
binding by subtracting the control sensorgram (Fc2) from the HveAt
surface sensorgram (Fc1). Model curve fitting was done with a 1:1
Langmuir binding model with drifting baseline. This is the simplest
model for the interaction between receptor and ligand; it follows
the equation A + B Molecular weight and oligomeric state analysis.
To calculate
kinetic and affinity constants, the oligomeric state and accurate
molecular weight of the protein flowing over the sensor chip surface
need to be known. Two observations suggest that full-length gD is a
dimer: (i) preparations of immunoaffinity-purified full-length gD1
contain a dimer that is resistant to sodium dodecyl sulfate (SDS) and
reducing conditions (8) and (ii) gD can be detected as
dimers and trimers in virions (14). On native
SDS-polyacrylamide gel, gDt is present primarily as a monomer, although
various amounts of dimer were routinely observed (33, 36).
However, the native gel system may not have given an accurate
representation of the amount of dimer present because the
small amounts of SDS present in the system probably disrupted a portion
of the noncovalently bound dimer. In this study we estimated the
molecular size of gDt by mass spectrometry, gel filtration
chromatography, and STEM.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Examination of the Kinetics of Herpes Simplex Virus Glycoprotein
D Binding to the Herpesvirus Entry Mediator, Using Surface
Plasmon Resonance
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
6 M and
gD2(306t) had a KD of 1.5 × 106 M. The interaction between gDt and HveAt fits a 1:1
Langmuir binding model, i.e., two dimers of HveAt may act as one
binding unit to interact with one dimer of gDt as the second binding
unit. A gD variant lacking all signals for N-linked oligosaccharides had an affinity for HveAt similar to that of gD1(306t). A variant lacking the bond from cysteine 1 to cysteine 5 had an affinity for
HveAt that did not differ from that of the wild type. However, variants
with double cysteine mutations that eliminated either of the other two
disulfide bonds showed decreased affinity for HveAt. This
result suggests that two of the three disulfide bonds of gD are
important for receptor binding. Four nonfunctional gDt variants, each
representing one functional domain of gD, were also studied. Mutations
in functional regions I and II drastically decreased the affinity of
gDt for HveAt. Surprisingly, a variant with an insertion
in functional region III had a wild-type level of affinity for HveAt,
suggesting that this domain may function in virus entry at a step other
than receptor binding. A variant with a deletion in
functional region IV [gD1(
290-299t)] exhibited a 100-fold
enhancement in affinity for HveAt (KD = 3.3 × 108 M) due mainly to a 40-fold increase in
its kinetic on rate. This agrees with the results of other studies
showing the enhanced ability of gD1(290-299t) to block infection.
Interestingly, all the variants with decreased affinities for HveAt
exhibited decreased kinetic on rates but only minor changes in their
kinetic off rates. The results suggest that once the
complex between gDt and HveAt forms, its stability is unaffected by a
variety of changes in gD.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
290-299t), was able to bind to HveAt by
ELISA and block virus entry 100-fold better than the wild-type protein,
gD1(306t) (33, 50). While ELISA has yielded valuable
information on the conditions and specificity of the gD-HveA
interaction, we are unable to determine the kinetics of binding by this
technique. Furthermore, ELISA detects only those molecules that remain
bound to receptor after multiple washes.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
80°C.
290-299t) was
eluted from the Superdex 200 column. The molecular weights of the
proteins were calculated from a standard plot according to the
directions of the calibration kits.
AB. The rate of association
(kon) is measured from the forward reaction, while koff is measured from the reverse reaction
(2).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
290-299t)] were used (Fig.
1; Table
1). The molecular mass of gD1 (QAAt)
determined by mass spectrometry is close to the formula mass. The mass
spectrometry values for gD1 (306t), gD2 (306t), and
gD1 (290-299t) are each 2,000 to 3,000 Da higher than that for
gD1 (QAAt). This is consistent with the addition of two to three
N-CHO, each with a mass of 1,000 Da (34). For all four
proteins, the masses estimated by gel filtration were approximately
twice those obtained by mass spectrometry. The gel filtration
experiment was repeated multiple times for gD1 (306t) and
gD1 (290-299t). Because mass spectrometry disrupts protein
oligomers that are not covalently bound, the gel filtration data
indicated that each protein exists as a noncovalent dimer in solution.
To confirm this, samples of gD1 (306t), gD2 (306t), and
gD1 (QAAt) were submitted for STEM analysis. Individual molecules of
protein were directly viewed by this technique, and masses were
estimated by comparison to a standard. Because this technique does not
disrupt noncovalent interactions, it is useful in determining the
oligomeric state of individual molecules (45, 47). The range
of masses for gD1 (306t), gD2 (306t), and gD1 (QAAt) was plotted on a histogram (Fig. 2), and the
average masses are reported in Table 1. These values agree with the gel
filtration values. Because mass spectrometry is most accurate for mass
determination, protein concentrations were calculated with dimeric
sizes for variants based on these values [e.g., 74,400 Da for
gD1 (306t), which is equal to two times the mass spectrometry
value].
View larger version (13K):
[in a new window]
FIG. 1.
Schematic representation of truncated HSV gD produced
by baculovirus-infected insect cells (33, 36). The
protein has the honeybee melittin signal peptide (hatched box) in
place of the wild-type gD signal for efficient translocation into the
lumen of the endoplasmic reticulum, two extra amino acids (D and P) at
the N terminus, and a histidine tag at the C terminus (33, 36,
44). The protein was truncated prior to the transmembrane region
and has three N-CHO (balloons). The disulfide bonds are indicated by
dashed lines. The functional regions of gD encompass the following
amino acids: 27 to 43 for region I, 126 to 131 for region II, 225 to
246 for region III, and 277 to 300 for region IV (5).
TABLE 1.
Summary of mass analysis data
View larger version (21K):
[in a new window]
FIG. 2.
STEM. Individual molecules of protein were directly
viewed, and molecular masses were calculated by comparison to a tobacco
mosaic virus standard. The range of molecular masses for each protein
was plotted on a histogram as percentages of the total numbers of
particles counted. The average molecular masses are reported in Table
1.
Binding of gD1 (306t) and gD1 (290-299t) to immobilized
HveAt.
Previous studies showed that HveAt interacts with gDt
(25, 32, 50). To examine the kinetics of binding of gDt to
HveAt by SPR, HveAt was coupled to a sensor chip surface. Figure
3 shows an example of the raw sensorgram
data. In this experiment, gD1 (290-299t) at a concentration of
0.5 µM in PBS-T was allowed to flow over the chip surface. Fc1
contained the immobilized HveAt, while Fc2 was activated and blocked
without the addition of protein. In the initial 50 s of each
sensorgram, a buffer baseline was established. Sample was injected, and
the association of gDt and HveAt followed for 2 min. At 120 s, the
sample was replaced with buffer, and the dissociation of complex
followed for 2 min. The response on the y axis is measured
in response units. There was very little nonspecific binding of protein
to the activated and blocked carboxymethyl dextran surface on Fc2. In
the SPR experiments that followed, data from Fc2 were subtracted from
the data from Fc1 to correct for changes in bulk refractive index (RI;
represents a difference in solution composition between the buffer and
the protein solution) and nonspecific binding to the sensor chip
surface.
|
290-299t). The data were overlaid with the result of the
global fitting analysis and are shown in Fig. 4. The global fitting
analysis allows simultaneous fitting of both the association and
dissociation phases of the sensorgram to all curves in the working set.
This improves the robustness and stability of the fitting procedure
(2). The residual plots for the fitted data (the difference
between the experimental and fitted data [not shown]) were all within
±3 RU, and the chi-square values (a standard statistical measure of
the closeness of the fit; values below 10 are acceptable) were all below 4.
|
290-299t) for HveAt was
predicted to be 100-fold higher than the affinity of gD1 (306t) for
HveAt (50). The values for KD
calculated from the SPR data were 3.2 × 106 M for
gD1 (306t) and 3.3 × 108 M for
gD1 (290-299t). The 100-fold higher affinity (lower
KD) of gD1 (290-299t) for HveAt than that
of gD1 (306t) was due mainly to a 40-fold increase in
kon, the rate of formation of complex (Table
2). There was also a twofold decrease in
koff for gD1 (290-299t) compared to that
of gD1 (306t), which contributed to the increased affinity. Thus,
the SPR data agree with and explain the predictions from ELISA data.
|
290-299t)] (Table 2).
Equilibrium binding of gD1 (306t) and gD1 (290-299t) as
determined by Scatchard analysis.
The binding assays of
gD1 (306t) and gD1 (290-299t) to HveAt were repeated under
equilibrium conditions to confirm the affinity constants calculated by
global analysis. Binding of gD1 (306t) to HveAt was monitored for 10 to 15 min, until equilibrium was reached. Binding of
gD1 (290-299t) to HveAt was monitored for 10 min. Dissociation
for each was monitored for 2 min. The flow rate for the experiment was
5 µl/min. We tested a range of concentrations of each protein from 5 to 0.15 µM for gD1 (306t) and from 1 to 0.03 µM for
gD1 (290-299t). Equilibrium was reached when the association
portion of the curve flattened (Fig. 5A
and B). Scatchard analysis of the data (Fig. 5C and D) yielded values
for KD of 2.3 × 106 M and
4.2 × 108 M for gD1 (306t) and
gD1 (290-299t), respectively. These values are essentially the
same as those obtained from the global analysis (Table 2).
|
Binding of HveAt to immobilized gD1 (306t) and
gD1 (290-299t).
In order to determine if the kinetics of
binding between gDt and HveAt were dependent on the orientation of the
complex, the reverse experiment was performed. Two chips were made
analogously to the HveAt chip surface, one with gD1 (306t)
covalently linked and the other with gD1 (290-299t) covalently
linked. HveAt was allowed to flow over each chip individually. The
signal obtained from the gD1 (306t) chip surface was very noisy
(data not shown). Analysis of the gD1 (306t) data, however,
indicated that the binding kinetics for the formation of complex were
the same as when the molecules were in the reverse orientation (Table
2). In contrast, HveAt bound to and dissociated from the immobilized
gD1 (290-299t), but the data did not fit any of the available
models (data not shown). Thus, the quality of data obtained was higher
when HveAt was immobilized on the chip surface than when either
gD1 (306t) or gD1 (290-299t) was immobilized. We previously
found that chemical modification of gDt alters its biological activity
(30, 51). Therefore, HveAt is more amenable to covalent
linkage through primary amines than gDt.
Binding of gDt variants to immobilized HveAt. Our approach to define the relationship between the structure of gD and its function has been to generate a panel of mutations within gD. With the discovery of HveA, a cellular receptor for HSV that binds gD, these variants become important once again to help determine the mechanism by which virus binds HveA and enters cells.
(i) gD1 versus gD2. Whitbeck et al. (50) reported that gD2 (306t) bound to HveAt with an affinity similar to that of gD1 (306t) as measured by ELISA. The kinetics of binding of gD2 (306t) to HveAt were compared with those of gD1 (306t) to see if differences could be detected in kon and/or koff. The fitted data resulted in values for kon, koff, and KD for gD2 (306t) that are essentially the same as those for gD1 (306t) (Fig. 6A; Table 2).
|
(ii) N-CHO. Glycosylation of gD does not play a role in the binding of gDt to HveAt as measured by ELISA (50), and the N-CHO on gD are not important for the infectivity of the virus (39, 43). gD1 (QAAt) (36), has three mutations which ablate the signals for addition of all three N-CHO. As expected, gD1 (QAAt) bound to HveAt with the same affinity as gD1 (306t) (Fig. 6B; Table 2). In addition, the absence of N-CHO did not alter kon or koff.
(iii) Disulfide bonds. gD has three disulfide bonds that are necessary for maintaining the native structure and stability of the molecule; the pairings consist of Cys 1 to 5, Cys 2 to 6, and Cys 3 to 4 (Fig. 1) (23). We previously found that double cysteine mutations in gD that removed any one disulfide bond resulted in proteins that were able to bind conformation-dependent monoclonal antibodies (MAbs) and that were also able to complement a gD null virus, but only in a temperature-sensitive manner (23). Three cysteine variants were cloned into a baculovirus expression system. Each variant contained mutations of two cysteine residues that eliminated one disulfide bond in gD (Fig. 1). First, the variants were tested for their abilities to bind conformation-dependent MAbs by native Western and dot blot analyses (data not shown). The binding profiles were the same as those measured with the full-length proteins (23). Next, ELISA was used to examine the abilities of the variants to bind HveAt both by ELISA (Fig. 7A) and SPR (Fig. 7B to D). By ELISA, gD1 (cys1,5) bound to HveAt as well as gD1 (306t) did (Fig. 7A). The other two variants, gD1 (cys2,6) and gD1 (cys3,4), bound to HveAt less well than gD1 (306t) did by ELISA. These results parallel the ability of the full-length variants to complement a gD null virus in trans (23). SPR experiments were done to quantitatively measure the magnitudes of binding differences among the variants. gD1 (cys1,5) had the highest affinity for HveAt, with values for kon, koff, and KD similar to those of gD1 (306t) (Table 2). gD1 (cys2,6) had an affinity for HveAt that was one-third that of gD1 (306t), due entirely to a lower kon (Table 2). Finally, the affinity of gD1 (cys3,4) for HveAt was one-fifth that of gD1 (306t) due to a corresponding decrease in kon (Table 2).
|
Binding of nonfunctional variants of gDt to HveAt.
gD has four
functional regions (I to IV) (Fig. 1) that are important for virus
entry (5, 28). Soluble forms of a representative nonfunctional variant from each region have been expressed in a
baculovirus expression system and previously characterized (31, 33). The functional region I variant, gD1 (
34t), did not
inhibit HSV plaque formation or cell-to-cell spread (33).
gD1 (34t) did not bind to HveAt by ELISA (Fig. 8A). By SPR at the
concentrations examined, the binding of this variant to HveAt was poor
(Fig. 8B) and the data did not fit a 1:1 model. Therefore, values for kon, koff, and
KD were not calculated for gD1 (34t). The
functional region II variant, gD1 (126t), exhibited poor
inhibition of plaque formation (33) and bound weakly to
HveAt by ELISA (Fig. 8A). The overall
affinity of gD1 (126t) for HveAt was seven times less than the
affinity of gD1 (306t) for HveAt due to a lower kon (Table 2). However, the
koff was similar to that of gD1 (306t), indicating that once the complex of gD1 (126t) and HveAt is
formed, it is as stable as the complex of gD1 (306t) and HveAt.
|
243t), bound to HveAt at
a level similar to that of gD1 (306t) by ELISA (Fig. 8A). The fitted
SPR data resulted in values for kon,
koff, and KD similar to
those of gD1 (306t) (Table 2). Although this mutant was
nonfunctional by complementation analysis (5), the soluble
protein was able to block HSV infection (33). Thus, the
binding data agree with the blocking data but differ from the results
of complementation analysis. In contrast, gD1 (290-299t), the
prototype variant from functional region IV, showed enhanced binding to
HveAt, as was characterized above (50).
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The mechanism of HSV entry is complicated, as it involves at least five envelope glycoproteins (41). A main function of gD is to interact with specific cellular receptors (3, 16, 17, 21, 25, 50). One of these, called herpesvirus entry mediator or HveA, is a member of the tumor necrosis factor receptor superfamily of membrane proteins and is found primarily on T cells and other cells of the immune system (25, 50). It is clear that (i) gDt and HveAt form a complex in solution (50), (ii) HveAt binds to gD on virions (32), and (iii) gDt interacts with immobilized HveAt (reference 50 and this report). The next step to complete the study of the interaction is a means to study the interaction of gD in the virus with HveA on the cell surface. The goal of this study was to document the binding capacity of gDt to HveAt and to begin to understand the characteristics of the interaction.
Kinetics of binding.
SPR was done to characterize the binding
between HveA, a receptor for HSV entry, and gD, the HSV
receptor-binding protein (25, 32, 50). We recently showed
that two forms of gDt appear to bind to HveAt with different affinities
(50). The first, gD1 (306t), is considered the wild-type
form of the protein, while the second, gD1 (290-299t), is a
functional region IV variant that shows enhanced inhibition of virus
entry on Vero cells (33) and enhanced binding to HveAt
(25, 50). The SPR data showed that the 100-fold increase in
the affinity of gD1 (290-299t) for HveAt was due primarily to a
40-fold increase in the kon (Table 3). Therefore, the rate of association
between gD1 (290-299t) and HveAt is higher than the rate of
association between gD1 (306t) and HveAt. However, the stability of
the gD1 (290-299t) HveAt complex once formed is similar to that
of the gD1 (306t)-HveAt complex (Table 3). Furthermore, the
difference in affinity between the two proteins was evident when
affinities were measured under both kinetic (short association time and
high flow rate) and equilibrium (long association time and low flow
rate) conditions of binding.
|
(i) Affinity of gD2 (306t) and gD1 (QAAt) for HveAt. Neither gD2 (306t) nor gD1 (QAAt) showed binding kinetics different from those of gD1 (306t) (Table 3), indicating that gDt from HSV-2 interacts with HveAt in the same fashion as gDt from HSV-1. Furthermore, N-CHO on gD1 do not play a role in this interaction.
(ii) Contribution of disulfide bonds to the binding of gDt to HveAt. Long et al. (23) showed that three variants of gD, each lacking one disulfide bond, can fold into a structure that resembles that of native gD. Of the three double cysteine variants, gD1(cys1,5) was clearly the least damaged, and this protein had the greatest capacity to complement the infectivity of a gD null virus. Though the three double-Cys variants did show some proper antigenic conformation, the ability of each protein to fold and to function was temperature sensitive. This emphasized the importance of all three bonds for the proper structure and function of gD. Here we produced truncated forms of the three double-Cys variant proteins. We took advantage of the fact that the insect cells produced the proteins at a (presumably) permissive temperature of 27°C. Antigenic analysis of the truncated proteins showed the same profiles seen with the full-length viral forms of the proteins. Binding to HveAt followed the trends seen in each full-length protein's ability to complement a gD null virus (23); gD1 (cys1,5) complements better than gD1 (cys2,6) and much better than gD1 (cys3,4). The affinity of gD1 (cys1,5) for HveAt was similar to that of gD1 (306t). However, the affinity of the gD1 (cys2,6) and gD1 (cys3,4) variants for HveAt were significantly lower. For both of these variants, the decrease in affinity was primarily due to a decrease in kon (Table 3). The koff values of all three variants were similar to that of wild-type gDt, indicating that the stability of the complex was not affected by the mutations removing any of the disulfide bonds. Thus, the disulfide bond between Cys 1 and 5 is not necessary for gDt binding to HveAt while the disulfide bonds between Cys 2 and 6 and Cys 3 and 4 are more important for the conformation of gDt that is necessary for binding to HveAt.
(iii) Affinity of nonfunctional variants for HveAt.
Four
functional regions in gD (Fig. 1) were identified by studying insertion
and deletion variants that retained antigenic conformation but lost the
ability to complement a gD null virus (5, 33). In the
binding experiments reported here, it appears that the mutations in
functional regions I and IV have the biggest effect on binding of gDt
to HveAt. The mutation within functional region I resulted in a loss of
HveAt binding ability. Nicola et al. (32) recently reported
that two groups of MAbs representing different epitopes on gD (sites Ib
and VII) are important for HveAt binding to gD in the virus. Antigenic
region VII (residues 11 to 19) (10, 20) is in close
proximity to functional region I. Thus, the gD1 (34t) insertion
may disrupt this region and diminish binding to HveAt. The functional
region IV variant exhibited enhanced HveAt binding ability (reference
50 and this report). This variant,
gD1 (290-299t), may be nonfunctional because of its high
kon, which leads to a high affinity for receptor
(50). One possibility is that the conformation of the
complex is altered such that later events in virus-cell fusion that
occur after the interaction of gD1 (290-299) with HveA are not
triggered. The characterization of binding of other functional region
IV variants as well as truncation variants of gDt to HveAt are
presented in a paper by Rux et al. (35).
126t), showed a marked
decrease in binding to HveAt by both ELISA and SPR, which perhaps accounts for its nonfunctional phenotype in complementation assays. In
contrast, the functional region III variant, gD1 (243t), showed a
wild-type level of binding to HveAt. This result led us to ask why the
full-length protein is nonfunctional in a complementation assay. Nicola
et al. (33) presented data consistent with the idea that gD
functions at more than one step in virus entry. Handler et al.
(13) showed that oligomeric associations among the
glycoproteins changed during HSV entry, and at least one study of HSV
entry suggested that individual glycoproteins function at distinct and different points in entry in a sequential, cascade-like mechanism (11). Perhaps the insertion in functional region III at
residue 243 alters the ability of gD to participate in a step in entry that occurs after receptor binding.
Johnson et al. (16) showed by Scatchard analysis that gD1
truncated at amino acid 275 from a mammalian expression system bound to
the cell surface with an affinity of 0.26 × 106 M. This value is intermediate between that of gD1 (306t) and gD1 (290-299t) (Table 2), possibly because (i) the gDt used for
our study was produced in insect cells while that used by Johnson et
al. (16) was from a mammalian system and/or (ii) Johnson et
al. looked at binding of gDt to the cell surface, which may contain
multiple receptors for gD (25, 50). In this study, we looked
at direct binding between gDt and HveAt.
Significance of a higher kon with little to
no change in koff.
Three recent reports
look at the effects of point mutations on kon
and koff in the binding of various proteins to
receptors (6, 26, 48). Morton et al. (26) looked
at mutations in human interleukin 5 (hIL-5) believed to be contact
residues in the binding of hIL-5 to the 
chain of its receptor. They
reported up to 5-fold changes in kon and up to
10-fold changes in koff. Some of the mutations
affected both kon and
koff significantly. Cunningham and Wells
(6) looked at the 31 side chains of residues buried at the
interface between human growth hormone and the extracellular binding
domain of its receptor. They found that mutations in one-quarter of the buried interface side chains led to a decrease in the
koff, which stabilized the interaction between
the two proteins. Up to 25-fold increases in
koff were measured for some of the mutations, while the largest change in kon was 3-fold. Wang
et al. (48) looked at mutations in proposed binding residues
of hIL-4 with its cellular receptor. The mutations helped to define a
koff epitope, i.e., residues that are involved
in stabilizing the formed complex. Mutations that reduced the charge of
this epitope increased koff by up to
360- fold, while the largest change in kon
was a 5-fold decrease.
Oligomerization of gDt and the gDt-HveAt complex.
A number of
reports have indicated that full-length gD1 may oligomerize into dimers
or trimers in the virion, on the surfaces of HSV-infected cells, and
when purified (8, 14). There are no reports that gD from
HSV-2 forms dimers. Here we show that both soluble gD1 and gD2 form
noncovalent dimers in solution. Furthermore, N-CHO on gD1 do not play a
role in dimer formation. We first used mass spectrometry to obtain
accurate molecular masses for the monomeric forms of the proteins
(34) and then gel filtration chromatography and STEM to show
that gD1 (306t), gD2 (306t), gD1 (QAAt), and
gD1 (290-299t) (50) form dimers in solution.
290-299t)-HveAt complex is 1:2. If gDt interacts in the complex as a dimer, then HveAt interacts in the complex as two dimers.
The fact that the complex fits a 1:1 binding model suggests that the
two dimers of HveAt act as one binding unit and that the gDt dimer acts
as one binding unit. Thus, there is a 1:1 interaction between binding
units. Alternatively, even though gDt is present as a dimer in
solution, it may dissociate and interact as a monomer with one dimer of
HveAt to yield a 1:1 binding interaction. When other binding models
depicting more complicated binding schemes were tried, none fit the
data as well as the 1:1 Langmuir binding model.
It is possible to determine the stoichiometry of the gDt-HveAt complex
being formed on the surface of the chip, provided that all of the
immobilized HveAt is active (2). However, in our system, not
all of the immobilized HveAt was active. Therefore, various methods to
uniformly orient the coupling of HveAt to the chip surface are being
investigated. Analytical ultracentrifugation experiments are also in
progress to determine the stoichiometries of the different gDt-HveAt
complexes.
| |
ACKNOWLEDGMENTS |
|---|
This investigation was supported by Public Health Service grants AI-18289 from the National Institute of Allergy and Infectious Diseases and NS-36731 and NS-30606 from the National Institute of Neurological Diseases and Stroke. S.H.W. and A.H.R. received support from Public Health Service grant AI-07324, and A.V.N. received support from Public Health Service grant AI-07325. We thank the Schools of Dental and Veterinary Medicine at the University of Pennsylvania for supplying funds for the purchase of a BIACORE X.
We thank John D. Lambris and William T. Moore of the Protein Chemistry Laboratory of the School of Medicine, University of Pennsylvania, for the mass spectrometry data. We thank Beth Lin for STEM sample preparation and Martha Simon and Joseph Wall of the Biology Department at the Brookhaven National Laboratory, Upton, N.Y., for collecting and analyzing the STEM data. (The Brookhaven STEM is supported by NIH grant P41-RR01777 and by the U.S. Department of Energy.) We thank Gabriela Canziani, manager of the Biosensor/Interaction Analysis Core Facility, and Irwin Chaiken and Sheng-jiun Wu at the School of Medicine of the University of Pennsylvania for help with SPR training and data analysis. We also thank Claude Krummenacher for a critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology, School of Dental Medicine, University of Pennsylvania, 4010 Locust St., Philadelphia, PA 19104-6002. Phone: (215) 898-6553. Fax: (215) 898-8385. E-mail: willis{at}biochem.dental.upenn.edu.
Present address: Institute for Biochemistry, Swiss Federal
Institute of Technology, 8092 Zurich, Switzerland.
Present address: IGP, Northwestern University Medical School,
Chicago, IL 60611.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Baker, S. J., and E. P. Reddy. 1996. Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 12:1-9[Medline]. |
| 2. | Biacore, A. B. 1997. In BIAevaluation software handbook, version 3.0. Biacore AB, Uppsala, Sweden. |
| 3. |
Campadelli-Fiume, G.,
M. Arsenakis,
F. Farabegoli, and B. Roizman.
1988.
Entry of herpes simplex virus 1 in BJ cells that constitutively express viral glycoprotein D is by endocytosis and results in degradation of the virus.
J. Virol.
62:159-167 |
| 4. |
Casasnovas, J. M., and T. A. Springer.
1995.
Kinetics and thermodynamics of virus binding to receptor.
J. Biol. Chem.
270:13216-13224 |
| 5. |
Chiang, H.-Y.,
G. H. Cohen, and R. J. Eisenberg.
1994.
Identification of functional regions of herpes simplex virus glycoprotein gD by using linker-insertion mutagenesis.
J. Virol.
68:2529-2543 |
| 6. | Cunningham, B. C., and J. A. Wells. 1993. Comparison of a structural and a functional epitope. J. Mol. Biol. 234:554-563[Medline]. |
| 7. | Eisenberg, R. J., D. Long, D. L. Sodora, H.-Y. Chiang, W. C. Wilcox, W. R. Abrams, M. I. Muggeridge, and G. H. Cohen. 1994. Structure and function of glycoprotein D of herpes simplex virus, p. 43-65. In Y. Becker, and G. Darai (ed.), Frontiers in virology, vol. 3. Springer Verlag, Heidelberg, Germany. |
| 8. |
Eisenberg, R. J.,
M. Ponce de Leon,
L. Pereira,
D. Long, and G. H. Cohen.
1982.
Purification of glycoprotein gD of herpes simplex virus types 1 and 2 by use of monoclonal antibody.
J. Virol.
41:1099-1104 |
| 9. |
Feenstra, V.,
M. Hodaie, and D. C. Johnson.
1990.
Deletions in herpes simplex virus glycoprotein D define nonessential and essential domains.
J. Virol.
64:2096-2102 |
| 10. | Friedman, H. M., G. H. Cohen, R. J. Eisenberg, C. A. Seidel, and D. B. Cines. 1984. Glycoprotein C of herpes simplex virus 1 acts as a receptor for the C3b complement component on infected cells. Nature (London) 309:633-635[Medline]. |
| 11. |
Fuller, A. O., and W. C. Lee.
1992.
Herpes simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gD and gH in penetration.
J. Virol.
66:5002-5012 |
| 12. | Geraghty, R. J., C. Krummenacher, R. J. Eisenberg, G. H. Cohen, and P. G. Spear. Entry of alphaherpesviruses mediated by poliovirus receptor related protein 1 and poliovirus receptor. Science, in press. |
| 13. | Handler, C. G., G. H. Cohen, and R. J. Eisenberg. 1996. Cross-linking of glycoprotein oligomers during herpes simplex virus type 1 entry. J. Virol. 70:6076-6082[Abstract]. |
| 14. | Handler, C. G., R. J. Eisenberg, and G. H. Cohen. 1996. Oligomeric structure of glycoproteins in herpes simplex virus type 1. J. Virol. 70:6067-6075[Abstract]. |
| 15. | Hillenkamp, F., M. Karas, R. C. Beavis, and B. T. Chait. 1991. Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers. Anal. Chem. 63:1193A-1203A[Medline]. |
| 16. |
Johnson, D. C.,
R. L. Burke, and T. Gregory.
1990.
Soluble forms of herpes simplex virus glycoprotein D bind to a limited number of cell surface receptors and inhibit virus entry into cells.
J. Virol.
64:2569-2576 |
| 17. |
Johnson, D. C., and M. W. Ligas.
1988.
Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors.
J. Virol.
62:4605-4612 |
| 18. | Karlsson, R., A. Michaelsson, and A. Mattson. 1991. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J. Immunol. Methods 145:229-240[Medline]. |
| 19. | Krummenacher, C., A. V. Nicola, J. C. Whitbeck, H. Lou, W. Hou, J. D. Lambris, R. J. Geraghty, P. G. Spear, G. H. Cohen, and R. J. Eisenberg. Herpes simplex virus glycoprotein D can bind to poliovirus receptor related protein 1 (PRR1/HveC) or herpes virus entry mediator (HVEM/HveA), two structurally unrelated mediators of virus entry. Submitted for publication. |
| 20. | Lasonder, E., G. A. Schellenkens, D. G. Koedijk, R. A. Damhof, S. Welling-Wester, M. Feijlbrief, A. J. Scheffer, and G. W. Welling. 1996. Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance. Eur. J. Biochem. 240:209-214[Medline]. |
| 21. |
Lee, W. C., and A. O. Fuller.
1993.
Herpes simplex virus type 1 and pseudorabies virus bind to a common saturable receptor on Vero cells that is not heparan sulfate.
J. Virol.
67:5088-5097 |
| 22. |
Long, D.,
G. H. Cohen,
M. I. Muggeridge, and R. J. Eisenberg.
1990.
Cysteine mutants of herpes simplex virus type 1 glycoprotein D exhibit temperature-sensitive properties in structure and function.
J. Virol.
64:5542-5552 |
| 23. |
Long, D.,
W. C. Wilcox,
W. R. Abrams,
G. H. Cohen, and R. J. Eisenberg.
1992.
Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2.
J. Virol.
66:6668-6685 |
| 24. |
Mauri, D. N.,
R. Ebner,
K. D. Kochel,
R. I. Montgomery,
T. C. Cheung,
G.-L. Yu,
M. Murphy,
R. J. Eisenberg,
G. H. Cohen,
P. G. Spear, and C. F. Ware.
1998.
LIGHT, a new member of the TNF superfamily, and lymphotoxin (LT) are ligands for herpesvirus entry mediator (HVEM).
Immunity
8:21-30[Medline].
|
| 25. | Montgomery, R. I., M. S. Warner, B. J. Lum, and P. G. Spear. 1996. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87:427-436[Medline]. |
| 26. |
Morton, T. A.,
J. Li,
R. Cook, and I. M. Chaiken.
1995.
Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor chain recognition.
Proc. Natl. Acad. Sci. USA
92:10879-10883 |
| 27. | Muggeridge, M. I., H.-Y. Chiang, G. H. Cohen, and R. J. Eisenberg. 1992. Mapping of a functional site on herpes simplex virus glycoprotein D. J. Cell. Biochem. Suppl. 16C:132. |
| 28. |
Muggeridge, M. I.,
W. C. Wilcox,
G. H. Cohen, and R. J. Eisenberg.
1990.
Identification of a site on herpes simplex virus type 1 gD that is essential for infectivity.
J. Virol.
64:3617-3626 |
| 29. | Myszka, D. G. 1997. Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. Curr. Opin. Biotechnol. 8:50-57[Medline]. |
| 30. | Nicola, A. V., R. J. Eisenberg, and G. H. Cohen. 1997. Unpublished data. |
| 31. | Nicola, A. V., C. Peng, H. Lou, G. H. Cohen, and R. J. Eisenberg. 1997. Antigenic structure of soluble herpes simplex virus glycoprotein D correlates with inhibition of HSV infection. J. Virol. 71:2940-2946[Abstract]. |
| 32. |
Nicola, A. V.,
M. Ponce de Leon,
R. Xu,
W. Hou,
J. C. Whitbeck,
C. Krummenacher,
R. I. Montgomery,
P. G. Spear,
R. J. Eisenberg, and G. H. Cohen.
1998.
Monoclonal antibodies to distinct sites on the herpes simplex virus (HSV) glycoprotein D block HSV binding to HVEM.
J. Virol.
72:3595-3601 |
| 33. | Nicola, A. V., S. H. Willis, N. N. Naidoo, R. J. Eisenberg, and G. H. Cohen. 1996. Structure-function analysis of soluble forms of herpes simplex virus glycoprotein D. J. Virol. 70:3815-3822[Abstract]. |
| 34. |
Rux, A. H.,
W. T. Moore,
J. D. Lambris,
W. R. Abrams,
C. Peng,
H. M. Friedman,
G. H. Cohen, and R. J. Eisenberg.
1996.
Disulfide bond structure determination and biochemical analysis of glycoprotein C from herpes simplex virus.
J. Virol.
70:5455-5465 |
| 35. | Rux, A. H., S. H. Willis, A. V. Nicola, W. Hou, H. Lou, C. Peng, G. H. Cohen, and R. J. Eisenberg. Functional region IV of glycoprotein D from herpes simplex virus modulates glycoprotein binding to the herpes simplex virus entry mediator (HVEM/HveA). Submitted for publication. |
| 36. |
Sisk, W. P.,
J. D. Bradley,
R. J. Leipold,
A. M. Stoltzfus,
M. Ponce de Leon,
M. Hilf,
C. Peng,
G. H. Cohen, and R. J. Eisenberg.
1994.
High-level expression and purification of secreted forms of herpes simplex virus type 1 glycoprotein gD synthesized by baculovirus-infected insect cells.
J. Virol.
68:766-775 |
| 37. | Smith, C. A., T. Farrah, and R. G. Goodwin. 1994. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76:959-962[Medline]. |
| 38. |
Sodora, D. L.,
G. H. Cohen, and R. J. Eisenberg.
1989.
Influence of asparagine-linked oligosaccharides on antigenicity, processing, and cell surface expression of herpes simplex virus type 1 glycoprotein D.
J. Virol.
63:5184-5193 |
| 39. |
Sodora, D. L.,
G. H. Cohen,
M. I. Muggeridge, and R. J. Eisenberg.
1991.
Absence of asparagine-linked oligosaccharides from glycoprotein D of herpes simplex virus type 1 results in a structurally altered but biologically active protein.
J. Virol.
65:4424-4431 |
| 40. |
Sodora, D. L.,
R. J. Eisenberg, and G. H. Cohen.
1991.
Characterization of a recombinant herpes simplex virus which expresses a glycoprotein D lacking asparagine-linked oligosaccharides.
J. Virol.
65:4432-4441 |
| 41. | Spear, P. G. 1993. Membrane fusion induced by herpes simplex virus, p. 201-232. In J. Bentz (ed.), Viral fusion mechanisms. CRC Press, Inc., Boca Raton, Fla. |
| 42. | Takemoto, D. K., J. J. Skehel, and D. C. Wiley. 1996. A surface plasmon resonance assay for the binding of influenza virus hemagglutinin to its sialic acid receptor. Virology 217:452-458[Medline]. |
| 43. | Tal-Singer, R., R. J. Eisenberg, T. Valyi-Nagy, N. W. Fraser, and G. H. Cohen. 1994. N-linked oligosaccharides on herpes simplex virus glycoprotein gD are not essential for establishment of viral latency or reactivation in the mouse eye model. Virology 202:1050-1053[Medline]. |
| 44. | Tessier, D. C., D. Y. Thomas, H. E. Khouri, F. Laliberte, and T. Vernet. 1991. Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. Gene 98:177-183[Medline]. |
| 45. |
Thomas, D. J.,
J. S. Wall,
J. F. Hainfield,
M. Kaczorek,
R. P. Booy,
B. L. Trus,
F. A. Eiserling, and A. C. Stevens.
1991.
gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains.
J. Virol.
65:3797-3803 |
| 46. | Wall, J. S. 1979. Biological scanning transmission electron microscopy, p. 333-342. In J. J. Hren, J. I. Goldstein, and D. C. Joy (ed.), Introduction to analytical electron microscopy. Plenum Publishing Corp., New York, N.Y. |
| 47. | Wall, J. S., and J. F. Hainfeld. 1986. Mass mapping with the scanning transmission electron microscope. Annu. Rev. Biophys. Chem. 15:355-376[Medline]. |
| 48. |
Wang, Y.,
B.-J. Shen, and W. Sebalk.
1997.
A mixed-charge pair in human interleukin 4 dominates high-affinity interaction with the receptor chain.
Proc. Natl. Acad. Sci. USA
94:1657-1662 |
| 49. | Warner, M. S., W. Martinez, R. J. Geraghty, R. I. Montgomery, J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear. A cell surface protein with herpesvirus entry activity (HveB) confers susceptibility to infection by herpes simplex virus type 2, mutants of herpes simplex virus type 1 and pseudorabies virus. Virology, in press. |
| 50. | Whitbeck, J. C., C. Peng, H. Lou, R. Xu, S. H. Willis, M. Ponce de Leon, T. Peng, A. V. Nicola, R. I. Montgomery, M. S. Warner, A. M. Soulika, L. A. Spruce, W. T. Moore, J. D. Lambris, P. G. Spear, G. H. Cohen, and R. J. Eisenberg. 1997. Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry. J. Virol. 71:6083-6093[Abstract]. |
| 51. | Whitbeck, J. C., C. P. Peng, G. H. Cohen, and R. J. Eisenberg. 1997. Unpublished data. |
| 52. | Williams, R. K., and S. E. Straus. 1997. Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans. J. Virol. 71:1375-1380[Abstract]. |
| 53. | Willis, S. H., C. Peng, M. Ponce de Leon, A. V. Nicola, A. H. Rux, G. H. Cohen, and R. J. Eisenberg. 1998. Expression and purification of secreted forms of HSV glycoproteins from baculovirus-infected insect cells. In S. M. Brown, and A. R. MacLean (ed.), Methods in molecular medicine, vol. 10, Herpes simplex virus protocols. Humana Press Inc., Totowa, N.J. |
J Virol, July 1998, p. 5937-5947, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Bernstein, D. I., Cardin, R. D., Bravo, F. J., Strasser, J. E., Farley, N., Chalk, C., Lay, M., Fairman, J.
(2009). Potent Adjuvant Activity of Cationic Liposome-DNA Complexes for Genital Herpes Vaccines. CVI
16: 699-705
[Abstract] [Full Text] -
Mok, W., Stylianopoulos, T., Boucher, Y., Jain, R. K.
(2009). Mathematical Modeling of Herpes Simplex Virus Distribution in Solid Tumors: Implications for Cancer Gene Therapy. Clin. Cancer Res.
15: 2352-2360
[Abstract] [Full Text] -
Reske, A., Pollara, G., Krummenacher, C., Katz, D. R., Chain, B. M.
(2008). Glycoprotein-Dependent and TLR2-Independent Innate Immune Recognition of Herpes Simplex Virus-1 by Dendritic Cells. J. Immunol.
180: 7525-7536
[Abstract] [Full Text] -
Lazear, E., Carfi, A., Whitbeck, J. C., Cairns, T. M., Krummenacher, C., Cohen, G. H., Eisenberg, R. J.
(2008). Engineered Disulfide Bonds in Herpes Simplex Virus Type 1 gD Separate Receptor Binding from Fusion Initiation and Viral Entry. J. Virol.
82: 700-709
[Abstract] [Full Text] -
Aldaz-Carroll, L., Xiao, Y., Whitbeck, J. C., de Leon, M. P., Lou, H., Kim, M., Yu, J., Reinherz, E. L., Isaacs, S. N., Eisenberg, R. J., Cohen, G. H.
(2007). Major Neutralizing Sites on Vaccinia Virus Glycoprotein B5 Are Exposed Differently on Variola Virus Ortholog B6. J. Virol.
81: 8131-8139
[Abstract] [Full Text] -
Zhou, G., Roizman, B.
(2007). Separation of receptor-binding and profusogenic domains of glycoprotein D of herpes simplex virus 1 into distinct interacting proteins. Proc. Natl. Acad. Sci. USA
104: 4142-4146
[Abstract] [Full Text] -
Tengvall, S., Lundqvist, A., Eisenberg, R. J., Cohen, G. H., Harandi, A. M.
(2006). Mucosal Administration of CpG Oligodeoxynucleotide Elicits Strong CC and CXC Chemokine Responses in the Vagina and Serves as a Potent Th1-Tilting Adjuvant for Recombinant gD2 Protein Vaccination against Genital Herpes.. J. Virol.
80: 5283-5291
[Abstract] [Full Text] -
Aldaz-Carroll, L., Whitbeck, J. C., Ponce de Leon, M., Lou, H., Hirao, L., Isaacs, S. N., Moss, B., Eisenberg, R. J., Cohen, G. H.
(2005). Epitope-Mapping Studies Define Two Major Neutralization Sites on the Vaccinia Virus Extracellular Enveloped Virus Glycoprotein B5R. J. Virol.
79: 6260-6271
[Abstract] [Full Text] -
Connolly, S. A., Landsburg, D. J., Carfi, A., Whitbeck, J. C., Zuo, Y., Wiley, D. C., Cohen, G. H., Eisenberg, R. J.
(2005). Potential Nectin-1 Binding Site on Herpes Simplex Virus Glycoprotein D. J. Virol.
79: 1282-1295
[Abstract] [Full Text] -
Bernet, J., Mullick, J., Panse, Y., Parab, P. B., Sahu, A.
(2004). Kinetic Analysis of the Interactions between Vaccinia Virus Complement Control Protein and Human Complement Proteins C3b and C4b. J. Virol.
78: 9446-9457
[Abstract] [Full Text] -
Cheshenko, N., Keller, M. J., MasCasullo, V., Jarvis, G. A., Cheng, H., John, M., Li, J.-H., Hogarty, K., Anderson, R. A., Waller, D. P., Zaneveld, L. J. D., Profy, A. T., Klotman, M. E., Herold, B. C.
(2004). Candidate Topical Microbicides Bind Herpes Simplex Virus Glycoprotein B and Prevent Viral Entry and Cell-to-Cell Spread. Antimicrob. Agents Chemother.
48: 2025-2036
[Abstract] [Full Text] -
Bender, F. C., Whitbeck, J. C., Ponce de Leon, M., Lou, H., Eisenberg, R. J., Cohen, G. H.
(2003). Specific Association of Glycoprotein B with Lipid Rafts during Herpes Simplex Virus Entry. J. Virol.
77: 9542-9552
[Abstract] [Full Text] -
Milne, R. S. B., Hanna, S. L., Rux, A. H., Willis, S. H., Cohen, G. H., Eisenberg, R. J.
(2003). Function of Herpes Simplex Virus Type 1 gD Mutants with Different Receptor-Binding Affinities in Virus Entry and Fusion. J. Virol.
77: 8962-8972
[Abstract] [Full Text] -
Connolly, S. A., Landsburg, D. J., Carfi, A., Wiley, D. C., Cohen, G. H., Eisenberg, R. J.
(2003). Structure-Based Mutagenesis of Herpes Simplex Virus Glycoprotein D Defines Three Critical Regions at the gD-HveA/HVEM Binding Interface. J. Virol.
77: 8127-8140
[Abstract] [Full Text] -
Santoro, F., Greenstone, H. L., Insinga, A., Liszewski, M. K., Atkinson, J. P., Lusso, P., Berger, E. A.
(2003). Interaction of Glycoprotein H of Human Herpesvirus 6 with the Cellular Receptor CD46. J. Biol. Chem.
278: 25964-25969
[Abstract] [Full Text] -
Yu, X., Wang, Q.-Y., Guo, Y., Dolmer, K., Young, J. A. T., Gettins, P. G. W., Rong, L.
(2003). Kinetic Analysis of Binding Interaction between the Subgroup A Rous Sarcoma Virus Glycoprotein SU and Its Cognate Receptor Tva: Calcium Is Not Required for Ligand Binding. J. Virol.
77: 7517-7526
[Abstract] [Full Text] -
Cairns, T. M., Milne, R. S. B., Ponce-de-Leon, M., Tobin, D. K., Cohen, G. H., Eisenberg, R. J.
(2003). Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras. J. Virol.
77: 6731-6742
[Abstract] [Full Text] -
Collins, W. J., Johnson, D. C.
(2003). Herpes Simplex Virus gE/gI Expressed in Epithelial Cells Interferes with Cell-to-Cell Spread. J. Virol.
77: 2686-2695
[Abstract] [Full Text] -
Connolly, S. A., Landsburg, D. J., Carfi, A., Wiley, D. C., Eisenberg, R. J., Cohen, G. H.
(2002). Structure-Based Analysis of the Herpes Simplex Virus Glycoprotein D Binding Site Present on Herpesvirus Entry Mediator HveA (HVEM). J. Virol.
76: 10894-10904
[Abstract] [Full Text] -
Martinez, W. M., Spear, P. G.
(2002). Amino Acid Substitutions in the V Domain of Nectin-1 (HveC) That Impair Entry Activity for Herpes Simplex Virus Types 1 and 2 but Not for Pseudorabies Virus or Bovine Herpesvirus 1. J. Virol.
76: 7255-7262
[Abstract] [Full Text] -
Birkmann, A., Mahr, K., Ensser, A., Titgemeyer, F., Fleckenstein, B., Neipel, F.
(2001). Cell Surface Heparan Sulfate Is a Receptor for Human Herpesvirus 8 and Interacts with Envelope Glycoprotein K8.1. J. Virol.
75: 11583-11593
[Abstract] [Full Text] -
Whitbeck, J. C., Connolly, S. A., Willis, S. H., Hou, W., Krummenacher, C., Ponce de Leon, M., Lou, H., Baribaud, I., Eisenberg, R. J., Cohen, G. H.
(2001). Localization of the gD-Binding Region of the Human Herpes Simplex Virus Receptor, HveA. J. Virol.
75: 171-180
[Abstract] [Full Text] -
Rauch, D. A., Rodriguez, N., Roller, R. J.
(2000). Mutations in Herpes Simplex Virus Glycoprotein D Distinguish Entry of Free Virus from Cell-Cell Spread. J. Virol.
74: 11437-11446
[Abstract] [Full Text] -
Krummenacher, C., Baribaud, I., Ponce de Leon, M., Whitbeck, J. C., Lou, H., Cohen, G. H., Eisenberg, R. J.
(2000). Localization of a Binding Site for Herpes Simplex Virus Glycoprotein D on Herpesvirus Entry Mediator C by Using Antireceptor Monoclonal Antibodies. J. Virol.
74: 10863-10872
[Abstract] [Full Text] -
Muggeridge, M. I.
(2000). Characterization of cell-cell fusion mediated by herpes simplex virus 2 glycoproteins gB, gD, gH and gL in transfected cells. J. Gen. Virol.
81: 2017-2027
[Abstract] [Full Text] -
Whitbeck, J. C., Muggeridge, M. I., Rux, A. H., Hou, W., Krummenacher, C., Lou, H., van Geelen, A., Eisenberg, R. J., Cohen, G. H.
(1999). The Major Neutralizing Antigenic Site on Herpes Simplex Virus Glycoprotein D Overlaps a Receptor-Binding Domain. J. Virol.
73: 9879-9890
[Abstract] [Full Text] -
Krummenacher, C., Rux, A. H., Whitbeck, J. C., Ponce-de-Leon, M., Lou, H., Baribaud, I., Hou, W., Zou, C., Geraghty, R. J., Spear, P. G., Eisenberg, R. J., Cohen, G. H.
(1999). The First Immunoglobulin-Like Domain of HveC Is Sufficient To Bind Herpes Simplex Virus gD with Full Affinity, While the Third Domain Is Involved in Oligomerization of HveC. J. Virol.
73: 8127-8137
[Abstract] [Full Text] -
Pilling, A., Rosenberg, M. F., Willis, S. H., Jäger, J., Cohen, G. H., Eisenberg, R. J., Meredith, D. M., Holzenburg, A.
(1999). Three-Dimensional Structure of Herpes Simplex Virus Type 1 Glycoprotein D at 2.4-Nanometer Resolution. J. Virol.
73: 7830-7834
[Abstract] [Full Text] -
Sarrias, M. R., Whitbeck, J. C., Rooney, I., Spruce, L., Kay, B. K., Montgomery, R. I., Spear, P. G., Ware, C. F., Eisenberg, R. J., Cohen, G. H., Lambris, J. D.
(1999). Inhibition of Herpes Simplex Virus gD and Lymphotoxin-alpha Binding to HveA by Peptide Antagonists. J. Virol.
73: 5681-5687
[Abstract] [Full Text] -
Shukla, D., Rowe, C. L., Dong, Y., Racaniello, V. R., Spear, P. G.
(1999). The Murine Homolog (Mph) of Human Herpesvirus Entry Protein B (HveB) Mediates Entry of Pseudorabies Virus but Not Herpes Simplex Virus Types 1 and 2. J. Virol.
73: 4493-4497
[Abstract] [Full Text] -
Krummenacher, C., Nicola, A. V., Whitbeck, J. C., Lou, H., Hou, W., Lambris, J. D., Geraghty, R. J., Spear, P. G., Cohen, G. H., Eisenberg, R. J.
(1998). Herpes Simplex Virus Glycoprotein D Can Bind to Poliovirus Receptor-Related Protein 1 or Herpesvirus Entry Mediator, Two Structurally Unrelated Mediators of Virus Entry. J. Virol.
72: 7064-7074
[Abstract] [Full Text] -
Rux, A. H., Willis, S. H., Nicola, A. V., Hou, W., Peng, C., Lou, H., Cohen, G. H., Eisenberg, R. J.
(1998). Functional Region IV of Glycoprotein D from Herpes Simplex Virus Modulates Glycoprotein Binding to the Herpesvirus Entry Mediator. J. Virol.
72: 7091-7098
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»