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Journal of Virology, October 1999, p. 8127-8137, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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
Claude
Krummenacher,1,2,*
Ann
H.
Rux,1,2
J. Charles
Whitbeck,1,2
Manuel
Ponce-de-Leon,1,2
Huan
Lou,1,2
Isabelle
Baribaud,1,2
Wangfang
Hou,1,2
Changhua
Zou,1,2
Robert J.
Geraghty,3
Patricia G.
Spear,3
Roselyn J.
Eisenberg,2,4 and
Gary H.
Cohen1,2
Department of
Microbiology1 and Center for Oral Health
Research,2 School of Dental Medicine, and
School of Veterinary Medicine,4
University of Pennsylvania, Philadelphia, Pennsylvania 19104, and
Department of Microbiology-Immunology, Northwestern
University Medical School, Chicago, Illinois
606113
Received 20 May 1999/Accepted 6 July 1999
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ABSTRACT |
The human herpesvirus entry mediator C (HveC/PRR1) is a member of
the immunoglobulin family used as a cellular receptor by the
alphaherpesviruses herpes simplex virus (HSV), pseudorabies virus, and
bovine herpesvirus type 1. We previously demonstrated direct binding of
the purified HveC ectodomain to purified HSV type 1 (HSV-1) and HSV-2
glycoprotein D (gD). Here, using a baculovirus expression system, we
constructed and purified truncated forms of the receptor containing one
[HveC(143t)], two [HveC(245t)], or all three immunoglobulin-like
domains [HveC(346t)] of the extracellular region. All three
constructs were equally able to compete with HveC(346t) for gD binding.
The variable domain bound to virions and blocked HSV infection as well
as HveC(346t). Thus, all of the binding to the receptor occurs within
the first immunoglobulin-like domain, or V-domain, of HveC. These data
confirm and extend those of Cocchi et al. (F. Cocchi, M. Lopez, L. Menotti, M. Aoubala, P. Dubreuil, and G. Campadelli-Fiume, Proc. Natl.
Acad. Sci. USA 95:15700, 1998). Using biosensor analysis, we measured
the affinity of binding of gD from HSV strains KOS and rid1 to two
forms of HveC. Soluble gDs from the KOS strain of HSV-1 had the same
affinity for HveC(346t) and HveC(143t). The mutant gD(rid1t) had an
increased affinity for HveC(346t) and HveC(143t) due to a faster rate
of complex formation. Interestingly, we found that HveC(346t) was a
tetramer in solution, whereas HveC(143t) and HveC(245t) formed dimers,
suggesting a role for the third immunoglobulin-like domain of HveC in
oligomerization. In addition, the stoichiometry between gD and HveC
appeared to be influenced by the level of HveC oligomerization.
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INTRODUCTION |
Herpes simplex virus (HSV) utilizes
several of its 11 membrane glycoproteins during entry into mammalian
cells. Glycoprotein C (gC) and/or gB assure the initial attachment to
cell surface heparan sulfate proteoglycans but are not sufficient to
induce viral entry (23, 60). gD, gB, and the gH-gL complex
are required for fusion of the viral envelope with the cell plasma
membrane (17, 49). Binding of gD to a cell surface receptor
is a key step leading to membrane fusion, which could be inhibited by
soluble or membrane-bound gD (6, 17, 26, 27, 45).
Recently, several cellular receptors for HSV have been identified. HveA
(41) (previously called HVEM, ATAR [24], or
TR2 [31]) can be used as a receptor by most HSV-1 and
HSV-2 strains. HveB (PRR2) usage appears to be restricted to HSV-2,
some laboratory strains of HSV-1 (rid1 and ANG), and pseudorabies virus
(PRV) (15, 55). HveC (PRR1) allows entry of all HSV-1 and
HSV-2 strains tested to date, as well as PRV and bovine herpesvirus type 1 (BHV-1) (18, 34). Recently, Cocchi et al.
(10) isolated a splice variant of HveC, named HIgR, with an
extracellular domain and receptor properties identical to those of
HveC. In addition, a monoclonal antibody (MAb) raised against human
bladder carcinoma cells 5637 (MAb R1.302) (35), which
recognized both HveC and HIgR, could block HSV infection
(10).
Unlike HveA, which is a member of the tumor necrosis factor receptor
family and a receptor for lymphotoxin alpha and LIGHT (37,
41), HveB and HveC are members of the immunoglobulin (Ig)
superfamily (18, 55). They are closely related to the poliovirus receptor (PVR; CD155) (39), which does not
function as an HSV receptor but can be used by PRV and BHV-1 for entry into cells (18). CD155, HveB, and HveC are type I membrane
glycoproteins harboring three Ig-like domains (V-C2-C2) in their
extracellular portion (15, 34, 39). CD155, HveB, and HveC
mRNAs are ubiquitously expressed and can be alternately spliced to
yield proteins having different transmembrane and intracellular domains
(10, 15, 28). The cellular function of CD155 is not known,
although HveC and HveB appear to be involved in cell-cell interactions
via homophilic binding, both in humans and mice (1, 33, 52).
Cell surface Ig-like molecules are used by a large number of viruses to
enter cells. Among them are CD155 (PVR) used by poliovirus (39), CD4 by human immunodeficiency virus (HIV)
(12), CAR by coxsackie B virus and adenovirus
(4), ICAM-1 by rhinovirus (19, 51),
Bgp1a for mouse hepatitis virus (MHV) (57), or
NCAM for rabies virus (54). When characterized, the
virus-binding site has been localized to the most distal Ig domain of
these molecules (14, 16, 32, 38, 42). Evidence for the
involvement of the HveC variable domain (V-domain) in HSV infection has
also been recently presented (9). Truncated soluble forms of
HveA and HveC produced in baculovirus-infected insect cells were shown
to interact directly with HSV-gD by enzyme-linked immunosorbent assay
(ELISA), in solution, and on viral particles (29, 44, 56).
The binding of gD from different strains of HSV to either receptor
correlated exactly with the ability of those virus strains to use HveA
and/or HveC to enter cells (29, 41, 56). Using a soluble
form of HveC, we identified individual residues and antigenic regions
of gD that affected receptor binding both in vitro and on viral
particles (29, 44). In addition, soluble HveC was an
efficient inhibitor of viral infection of neuron-like cell lines in
culture such as IMR5 and SY5Y (18).
Recently, Cocchi et al. (9) reported evidence that the most
membrane-distal domain (V-domain) of HveC was sufficient to confer
susceptibility to HSV infection when expressed at the surface of
nonpermissive cells. However, the efficiency of infection appeared to
be markedly affected. The same study demonstrated that a soluble form
of the HveC V-domain linked to heterologous Ig-domains from the Fc part
of an Ig can block HSV infection of cultured cells and interact with a
soluble mutant form of gD (9, 45).
In order to extend the characterization of the direct interaction
between the receptor and gD, from structure and affinity standpoints,
we expressed smaller forms of soluble HveC. Here we show that a protein
containing just the single V-domain of HveC and a protein containing
the two distal domains of HveC are each able to bind to soluble gD as
efficiently as the whole HveC ectodomain. The HveC V-domain protein
bound to virus and was also able to block HSV infection of several
human neuron-like cell lines as efficiently as the full ectodomain.
Using surface plasmon resonance technology, we found that both soluble
receptors display similar affinity to several forms of HSV-1 gD.
Moreover, both the on and the off rates of gD-HveC complex formation
were very similar. Interestingly, the single V-domain protein and the
two-domain protein were dimers in solution, whereas the complete
ectodomain was a tetramer under the same conditions. This indicated the
possibility of several levels of oligomerization of HveC involving at
least the first and the third domains.
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MATERIALS AND METHODS |
Cells and viruses.
Spodoptera frugiperda Sf9 cells
(GIBCO BRL) were maintained in suspension in Sf900II medium or as
monolayer cultures in supplemented Grace's medium (GIBCO BRL) with
10% fetal calf serum (FCS). CHO M3A cells are derived from CHO-IE
8
cells expressing the -galactosidase gene under the control of the
viral ICP4 promoter (41, 53) and express constitutively the
full-length human HveC under the control of a cytomegalovirus promoter.
M3A cells were grown in HAM's F-12 medium supplemented with 10% FCS,
250 µg of G418 per ml, and 150 µg of puromycin per ml. IMR5 and
SY5Y (human neuroblastoma cell lines) were grown in Dulbecco modified
Eagle medium with 10% FCS. HSV-1 KOS tk12 and HSV-1 rid1 tk12
(41) were purified from infected Vero cells, and titers were
obtained on Vero cells (22).
Glycoproteins and antibodies.
Soluble glycoproteins such as
gD(306t) and gD(285t) were derived from HSV-1 strain KOS unless
otherwise noted; gD-2(306t) was from HSV-2 strain 333 and gD(rid1t)
from HSV-1 strain rid1. Construction and purification of these proteins
from baculovirus-infected cell supernatant was described elsewhere
(43, 45, 47, 48). Rabbit polyclonal serum R7 was raised
against HSV-2 gD purified from infected mammalian cells
(25). Anti tetra-His MAb was purchased from Qiagen, Inc.
Anti-HveC MAb R1.302 (35) was kindly provided by S. McClellan (Beckman Coulter).
Construction of recombinant baculoviruses and purification of
soluble receptors.
The strategy to generate soluble HveC(346t) was
described previously (29, 58) and was applied here to
generate all HveC constructs. Plasmid pBG38 was used as template for
PCR amplification by using the upstream primer (C5)
5'-GCGTGATCAGGTGGTCCAGGTGAACGACTCCATGTAT-3' and the
downstream primer
5'-CGGCCCGGGCTAATGATGATGATGATGATGCTGCACGTTGAGAGTGAGGCTTTCC-3' for HveC(245t) or
5'-CGGCCCGGGCTAATGATGATGATGATGATGCATCACCGTGAGATTGAGCTGGCTTTCT-3' for HveC(143t). The cloning strategy with the vector pVT-Bac was described earlier (29, 58), and plasmids pCK329 and pCK330, respectively, were generated and used to produce recombinant
baculoviruses bac-HveC(245t) and bac-HveC(143t). HveC(245t) contains
amino acids 31 to 245 from human HveC with an extra aspartic acid
residue at the N terminus and a 6-histidine tail at the C terminus of the protein added during the amplification and cloning process. HveC(143t) contains amino acids 31 to 143 of human HveC with the same
C- and N-terminal additions.
All soluble receptors, containing a C-terminal 6-histidine tag, were
purified by nickel affinity chromatography (Ni-NTA Superflow; Qiagen,
Inc.) as described previously (29, 56, 58). Purified soluble
HveC proteins were dialyzed against 100 mM sodium phosphate (pH
8.0)-150 mM NaCl and concentrated.
ELISA.
(i) The standard ELISA with immobilized receptor and
soluble gD was as described previously (29). (ii) For the
competition ELISA, HveC(346t) at 10 µg/ml in phosphate-buffered
saline (PBS) was adsorbed to microtiter plates for 2 h at room
temperature. Plates were washed with 0.1% Tween 20 in PBS and blocked
with 5% milk-0.2% Tween 20 in PBS (PBST-milk) for 1 h at room
temperature. Plates were then incubated overnight at 4°C with
PBST-milk containing a constant concentration of gD and variable
concentrations of soluble receptors as competitors. The plates were
washed, and the bound gD was detected with R7 antiserum (diluted
1:1,000 in PBST-milk) for 1 h, followed by the addition of goat
anti-rabbit IgG coupled to horseradish peroxidase (diluted 1:1,000 in
PBST-milk) for 30 min. The plate was washed with 20 mM citric acid (pH
4.5) prior to the addition of substrate (ABTS; Moss, Inc.). Absorbance was read at 405 nm.
Blocking assay.
Cells were grown to confluence in 96-well
plates in their respective medium and chilled for 15 min at 4°C prior
to the addition of virus. HSV-1 KOS tk12 or HSV-1 rid1 tk12 was
preincubated with soluble receptors at various concentrations in cold
medium containing 30 mM HEPES for 90 min at 4°C. Culture medium was
removed, and 100 µl of virus-containing medium was added
(multiplicity of infection of 1). Cells were then incubated at 37°C
for 5 to 6 h and lysed in NP-40 (0.5% final). Then, 50 µl of
cell lysate was mixed with an equal volume of -galactosidase
substrate (chlorophenol red--D-galactopyranoside). The
level of entry was monitored by reading the absorbance at 595 nm for 50 min to record the enzymatic activity, expressed as the change in
optical density per hour (
OD/h). Blocking activity of soluble
receptors is expressed as the percentage of virus entry into cells
under test conditions compared to viral infection in absence of
inhibitor (100%).
Gel filtration.
Purified proteins were diluted in PBS and
loaded onto a Superdex 200 column (Pharmacia HR 10/30) as described
previously (29).
Electrophoresis.
Nondenaturing and nonreducing
polyacrylamide gel electrophoresis (PAGE) has been described previously
(11). Proteins were separated on precast Tris-glycine gels
(Novex) by using 200 mM glycine-25 mM Tris base-0.1% sodium dodecyl
sulfate (SDS) as running buffer. Proteins were then visualized by
silver staining of the gel (Pharmacia Silver Stain Kit) or transferred
to nitrocellulose prior to antibody detection.
Binding of HveC to virus.
Soluble receptor (100 µg) was
mixed with 107 PFU of purified HSV-1 KOS (ca. 4 × 108 particles) or 107 PFU of purified HSV-1
rid1 (ca. 3 × 108 particles) in 150 µl of PBS for
90 min at 4°C. The virus was subjected to sedimentation through a
sucrose step gradient (10, 30, and 60%) for 4.5 h at
16,000 × g. The viral band was then collected and
analyzed by Western blotting as described previously (29,
44) by using anti-tetra-His (Qiagen, Inc.) and anti-VP5 antibodies (21).
Measurement of binding of gD to HveC with an optical
biosensor.
Biosensor experiments were carried out on a Biacore X
optical biosensor (Biacore AB) at 25°C according to the protocol
previously described (47, 59) but with the following
modifications. The running buffer was HBS-EP (10 mM HEPES, 150 mM NaCl,
3 mM EDTA, 0.005% polysorbate 20), pH 7.4. Approximately 1,600 response units (RU) of HveC(346t) or 300 RU of HveC(143t) were 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. Soluble gD was serially diluted in
HBS-EP. Each gD sample was injected for 2 min to monitor the association. The sample was then replaced by HBS-EP flow, and the
dissociation was monitored for 2 min. During the binding and dissociation phases of gD to HveC, 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 (five measurements/min). To regenerate the HveC
surface, brief pulses of 0.2 M Na2CO3 (pH 10)
were injected until the response signal returned to baseline.
Sensorgrams were corrected for nonspecific binding and refractive index
changes by subtracting the control sensorgram (Fc2) from the HveC
surface sensorgram (Fc1). Data were analyzed with BIAevaluation
software, version 3.0 (5). Model curve fitting was done by
using a 1:1 Langmuir interaction with drifting baseline. This models
the simple interaction between ligand (L) and receptor (R) as follows:
L + R 
LR. The rate of association (kon)
was measured from the forward reaction, and koff
was measured from the reverse reaction (5).
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RESULTS |
Production and characterization of baculovirus-expressed HveC
receptors.
Soluble receptors were purified from supernatant of
recombinant baculovirus-infected Sf9 cells. HveC(143t) contains the
N-terminal Ig-like V-domain, and HveC(245t) encompasses two N-terminal
domains (Fig. 1). Their properties were
compared to the previously described HveC(346t) (29), which
includes all three Ig-domains of HveC and ends just before the
transmembrane region (Fig. 1). As shown previously, HveC(346t) migrated
as a 45-kDa glycoprotein as seen by SDS-PAGE, although mass
spectrometry revealed a molecular mass of 40 kDa (29).
HveC(245t) migrated as a heterogeneous glycoprotein of 33 to 34 kDa
(Fig. 2A). Its molecular mass, determined
by mass spectrometry, was 28.0 kDa for the main species carrying three N-linked oligosaccharides (N-CHO); minor products with two or four
N-CHO were also present (data not shown). By SDS-PAGE, HveC(143t) appeared as three bands, probably representing the protein with one,
two, or three N-linked carbohydrates. The apparent sizes were 17, 19, and 22 kDa respectively (Fig. 2A). Mass spectrometric analysis of
HveC(143t) detected two major products of 14.5 and 15.6 kDa, as well as
a minor product of 16.7 kDa (data not shown). The observed increment of
size correlated with the addition of one N-linked carbohydrate chain in
insect cells (30, 46). In both cases, treatment of
HveC(245t) and HveC(143t) with glycopeptidase F yielded a single,
faster-migrating band (Fig. 2B). All N-CHO on HveC(143t) were resistant
to endoglycosidase H, whereas N-CHO on HveC(245t) were partially
sensitive (Fig. 2B). Thus, the endoglycosidase data suggest that the
oligosaccharide on Asn 202 present in HveC(245t) but not HveC(143t)
might not be processed from the high-mannose type to the complex type.
The presence of three glycosylated forms of HveC(143t) indicated that
three N-CHO consensus attachment sites were used. A fourth Asn residue
proposed as an N-CHO attachment site in the original sequence of
HveC/PRR1 (34) at position 82 in an Asn-Pro-Ser
pseudoconsensus site is probably not glycosylated.
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FIG. 1.
(A) Schematic representation of HSV receptors.
Full-length HveC is shown as a solid line with amino acids numbered
from the initial methionine. The open box indicates the HveC natural
signal peptide. (B) Schematic representation of gD constructs. gD from
HSV-1 KOS is represented with amino acids numbered from the N terminus
of the mature gD after cleavage of the gD signal peptide (shaded box).
Disulfide bonds are indicated by dotted lines. The black circles
represent putative N-linked carbohydrates. The hatched box represents
the mellitin signal peptide used in the baculovirus constructs.
Baculovirus-expressed proteins are truncated (t) at the indicated amino
acid prior to the transmembrane region (TMR). H6, six-histidine tag
added at the C terminus.
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FIG. 2.
Purified soluble receptors. Receptors purified from
recombinant baculovirus-infected Sf9 cell supernatants were separated
by SDS-PAGE. (A) Purified proteins were electrophoresed on a 12%
polyacrylamide gel under reducing and denaturing conditions and then
visualized by silver staining. The molecular mass markers are indicated
in kilodaltons. (B) Endoglycosidase digestions. HveC(245t) (lanes 1 to
4) and HveC(143t) (lanes 5 to 8) were subjected to digestion with
PNGase F (F) or endoglycosidase H (H) or were mock treated ( ) prior
to electrophoresis on a 16% acrylamide gel under reducing and
denaturing conditions. After the Western blotting, proteins were
detected with the anti-tetra-His MAb. (C) Native Western blot. Proteins
were run under nondenaturing and nonreducing conditions on a 12%
polyacrylamide gel and detected with MAb R1.302 (35).
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Detection of HveC truncations with MAbs.
The MAb R1.302 binds
to the single V-domain of HveC on cells and blocks virus entry (9,
10). We used this blocking MAb to probe a native Western blot of
the three soluble HveC proteins (Fig. 2C). The antibody detected each
form of the receptor, including all three glycosylated forms of
HveC(143t). Detection of HveC(143t) by MAb R1.302 was abolished when
PAGE was performed under reducing and denaturing conditions (data not
shown). This indicated that R1.302 recognized a nonlinear epitope on
HveC(143t) and that the purified proteins, including the one domain
HveC(143t), were correctly folded. We also tested the native
conformation of HveC truncations by ELISA (Fig.
3). As expected, all truncated forms of
HveC reacted similarly with the anti tetra-His antibody, indicating
that comparable amounts of proteins had been immobilized (Fig. 3A). In
the same setting, we used MAb R1.302 to detect a conformation-dependent epitope on truncated HveC immobilized on an ELISA plate (Fig. 3B). Both
HveC(346t) and HveC(245t) reacted with similar efficiency. In contrast,
HveC(143t) was less efficiently recognized by this MAb, suggesting a
loss of conformation of this small HveCt.
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FIG. 3.
Binding of MAbs and gD to HveC MAbs by ELISA. Detection
of immobilized truncated HveC proteins with anti-tetra-His Ig (Qiagen,
Inc.) (A) or R1.302 Ig (Beckman Coulter) (B). Bound immunoglobulins
were detected with horseradish peroxidase-conjugated anti-mouse IgG
secondary antibody and substrate. (C) Plates coated with HveC truncated
proteins were incubated with increasing concentrations of gD(306t) from
HSV-1 strain KOS. Bound gD was detected with R7 antiserum, followed by
peroxidase-conjugated secondary antibody and substrate. Absorbance was
read at 405 nm.
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Interaction of truncated HveC with gD by ELISA.
Since
HveC(346t) binds gD directly in an ELISA (29), we used this
method to evaluate gD binding to the three forms of HveC. Various
concentrations of soluble gD(306t) or gD(285t) (Fig. 1B) were incubated
with each form of HveC which has been immobilized on an ELISA plate.
Bound gD was then detected with polyclonal R7 serum. gD(306t) bound to
both HveC(346t) and HveC(245t) with similar efficiency, indicating that
the most C-terminal Ig domain is dispensable for the interaction of
HveC with gD (Fig. 3C). However gD(306t) bound less efficiently to
HveC(143t). The shorter gD(285t), which displays an enhanced binding
affinity for HveC over that of gD(306t) (see below and reference
47), also bound to HveC(143t) with reduced ability
compared to HveC(346t) and HveC(245t) (data not shown).
Binding of HveC truncations to gD in solution.
The ability of
each truncated HveC to bind gD by ELISA correlated with its capacity to
be recognized by the conformation-dependent blocking MAb R1.302.
Since heat-denatured HveC(346t) completely failed to bind gD by ELISA
(data not shown), the decreased binding ability of HveC(143t)
might
reflect a partial denaturation that could have occurred
during
purification or because of immobilization on the ELISA
plate. To
clarify this point, we performed a competition ELISA
where the binding
between gD and the three receptor forms occurred
in solution. In this
experiment HveC(346t) was immobilized on
the plate. Then a constant
amount of soluble gD(306t), corresponding
to the half-saturating
concentration for the bound HveC(346t),
was added in the absence or
presence of one of the soluble receptors
which acted as a competitor
for binding of gD (Fig.
4A). Soluble
HveC(346t), HveC(245t), or HveC(143t) competed the binding of
gD(306t)
to the immobilized HveC(346t) in a dose-dependent manner.
Both soluble
HveC(245t) and HveC(143t) were able to compete gD
binding nearly as
well as soluble HveC(346t) which competes with
itself on the plate for
gD binding.
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FIG. 4.
Competition ELISA. Plates were coated with HveC(346t)
and incubated with a constant amount of purified gD, together with
increasing concentrations of HveC truncated receptors as competitors.
Panels show 1 µM gD-1(306t) from HSV-1 KOS (A), 0.1 µM gD-1(rid1t)
(B), and 1 µM gD-2(306t) from HSV-2 strain 333 (C). gD bound to the
immobilized receptor was detected with R7 antiserum. Relative binding
is indicated as percentage of gD binding in the absence of soluble
receptor.
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HveC(346t) is also known to bind gD from HSV-2 and gD from the mutant
strain HSV-1 rid1 (
29,
56). The rid1 form of gD
has a point
mutation at position 27 (Q27P) (
13) which prevents
its
binding to HveA but enhances its binding to HveC in an ELISA
(
29,
56). Using the competition assay, we also tested the
binding of
soluble gD(rid1t) (Fig.
4B) and of gD-2(306t) from
HSV-2 strain 333 (Fig.
4C) to truncated HveC forms. For gD-1 rid1
and gD-2, all three
truncations competed the binding to HveC(346t)
with similar efficiency.
Thus, we conclude that soluble gD binds
HveC(143t) as well as it does
to the other two truncated forms
of HveC. Therefore, the poor binding
seen by direct ELISA (Fig.
3C) is due to changes in HveC(143t) that
occur as a result of
immobilization on the ELISA plate. Furthermore,
the data in Fig.
4 suggest that the affinity of all three HveC
truncations for
gD is
similar.
Affinity of binding between gD and HveC(346t) or HveC(143t).
Biosensor analysis was previously used to measure affinity between gD
mutant proteins and truncated form of the receptor HveA (47,
59). Here we used the same approach to determine the affinity of
gD for HveC(346t) and for the shorter form, HveC(143t). HveC was
coupled on the surface of one flow cell (Fc1) of a CM5 chip via primary
amines and the second flow cell (Fc2) was left bare. The Fc2 response
representing background sticking and bulk change of refractive index
was subtracted from the Fc1 response to obtain specific binding data
(Fig. 5). Serial dilutions of gD(306t)
(Fig. 5A and B), gD(285t) (Fig. 5C and D), and gD(rid1t) (Fig. 5E and
F) were flowed over chips carrying HveC(346t) (Fig. 5A, C, and E) or
HveC(143t) (Fig. 5B, D, and F). After a baseline was established, the
association of gD to immobilized HveC was monitored for 120 s.
Buffer was then substituted for gD solution, and the dissociation of
the complex was monitored for another 2 min. A global fit of the data
was obtained by using the BIAevaluation 3.0 software for a 1:1 Langmuir
model (Fig. 5). Kinetics values and affinity constants for each of the
pairs tested are summarized in Table 1.
In the case of HveC(346t) we calculated a dissociation constant
(KD) of 3.2 × 10
6 M for
gD(306t). The affinity of gD(285t) for HveC(346t) was enhanced more
than 80 times, mainly because of an increase of the on rate (kon). The association between HveC(346t) and
gD(rid1t) showed a KD of 1.7 × 107 M. The 20-fold increase in affinity compared to
gD(306t) was also due to a higher on rate.
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FIG. 5.
Analysis of gD binding to HveC in real time. HveC(346t)
(A, C, and E) or HveC(143t) (B, D, and F) were immobilized on a CM5
biosensor chip to 1,600 and 300 RU, respectively, in a Biacore X
instrument. Various concentrations of gD(306t) (A and B), gD(285t) (C
and D), and gD(rid1t) (E and F) were flowed over the chip for 2 min
(association) and then replaced by buffer for another 2 min
(dissociation). Sensorgrams of corrected data are represented after the
subtraction of signal from the control flow cell. Data points were
collected at 5 Hz but, for clarity, only one every 25 points is
represented here by a symbol. The solid line shows the best fit
obtained after global fitting with the BIAevaluation 3.0 software
(5).
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|
In the case of HveC(143t), the V-domain alone, the affinity for the
various forms of gD followed the same trend. gD(306t),
with a
KD of 1.2 × 10
6 M, had the
lowest affinity and gD(285t) (
KD = 3.5 × 10
8 M) showed the highest affinity. Again, the
variation in affinity
was caused primarily by changes in the rate of
complex formation.
Again, gD(rid1t) showed an intermediate affinity for
HveC(143t).
Both HveC(346t) and HveC(143t) displayed similar affinity for gD(285t)
or gD(rid1t), with no significant differences in either
on or off
rates, whereas gD(306t) appeared to have a slightly
higher affinity for
HveC(143t) than for
HveC(346t).
Blocking of HSV infection with soluble truncated forms of
HveC.
We previously showed that HveC(346t) blocked HSV-1 infection
of several human cell lines (18). We therefore examined the ability of HveC(245t) and HveC(143t) to block virus infection. In our
standard assay, purified HSV-1 KOS tk12 was preincubated with the three
soluble receptors prior to addition to target cells. M3A cells, derived
from CHO cells, express full-length human HveC constitutively.
Infection of M3A cells with HSV-1 KOS tk12 was efficiently blocked by
all three HveC truncations (Fig. 6A). No significant difference was detected in the ability of any of the truncations to inhibit infection.
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FIG. 6.
Blocking of HSV infection with soluble HveC truncations.
HSV-1 KOS tk12 was preincubated with variable concentrations of soluble
HveC truncations or BSA prior to addition to M3A (A), IMR5 (B), or SY5Y
(C) cells. Cells were lysed at 5.5 h postinfection, and the
-galactosidase activity was measured. A value of 100% of entry
corresponds to the -galactosidase activity induced after infection
with HSV-1 KOS tk12 at a similar multiplicity of infection (0.5 to 1 PFU/cell) in the absence of soluble inhibitor.
|
|
We extended this study to include the neuroblastoma cell lines IMR5 and
SY5Y. Infection of these cells has also been shown
to be inhibited by
soluble HveC(346t) (
18). Figure
6B and C
shows that
HveC(245t) and HveC(143t) were able to block HSV-1
infection of these
cells as efficiently as HveC(346t). Infection
of nondifferentiated NT-2
cells and HeLa cells could also be blocked
by all three soluble forms
of HveC with similar efficiency (data
not
shown).
Binding of soluble HveC to virion.
Binding of HveC(346t) to gD
on HSV-1 KOS virions was shown previously (29). The ability
of the smaller forms of HveC to block infection suggested that these
proteins also directly interacted with viral particles. To test this,
purified virions were preincubated with the soluble receptors prior to
sedimentation through a sucrose gradient. The presence of receptor in
the virus band collected from the gradient is indicative of direct
binding of receptor to virions. Western blot analysis of the viral band
showed that both HveC(346t) and HveC(143t) could be detected and thus
had bound to KOS viral particles (Fig.
7). The different glycosylated forms of
HveC(143t) could be detected. Similar amounts of each virus were
recovered and loaded onto the gel, as reflected by the amount of VP5
protein detected with the NC1 antibody (Fig. 7). Both HveC(346t) and
HveC(143t) were also bound to purified HSV rid1 particles in this assay
(data not shown).
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FIG. 7.
Binding of soluble HveC to HSV particles. Purified
HveC(346t) or HveC(143t) (100 µg) were incubated with 107
PFU of purified HSV-1 KOS for 90 min at 4°C. Viral particles were
then sedimented through a discontinuous sucrose gradient. The
virus-containing fraction was collected, concentrated, and analyzed by
Western blot for detection of HveC and virus. Receptors were detected
with anti-tetra-His antibody, and VP5 capsid protein was detected with
NC-1 polyclonal rabbit serum simultaneously.
|
|
Oligomerization of HveC(143t) or HveC(245t).
We previously
showed that HveC(346t) in solution appears as a high-molecular-mass
complex of 176 kDa, a size consistent with that of a tetramer
(29). Here gel filtration studies were performed with the
other two HveC truncations on a Superdex 200 size exclusion column.
HveC(245t), with a molecular mass determined by mass spectrometry of 28 kDa, elutes with an apparent size of 71 kDa (Fig.
8A), a finding consistent with the
formation of a dimer in solution. Similarly, the 15-kDa HveC(143t)
appears to form a 29-kDa dimer in solution (Fig. 8B). The difference in
oligomerization between HveC(346t), a tetramer (29), and
HveC(245t), a dimer, suggests that the third Ig-domain of HveC can
promote a higher level of oligomerization of HveC. gD(285t) eluted from
the column with an apparent size of 51 kDa, a result consistent with
the presence of a dimeric form of gD (Fig. 8A and B). We previously
observed that gD was essentially a dimer in solution and on virus
(22, 59).
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FIG. 8.
Gel filtration chromatography of HveC(245t) and
HveC(143t) alone or in a complex with gD(285t). Elution profiles of
HveC(245t) and HveC(143t) loaded at 33 and 66 µM respectively, on a
Superdex 200 column are shown in panels A and B as solid lines. An
elution profile of gD(285t) (26 µM) is shown as a dotted line in
panels A and B. Elution profiles of gD(285t)-HveC(245t) complex (C and
E) or gD(285t)-HveC(143t) complex (D and F) at the given ratios are
shown. Molecular sizes were determined by calibrating the column with
standards of proteins ranging from 13.7 to 669 kDa. The shaded area in
panel F indicates the fraction used for quantification of HveC(143t)
and gD(285t) in Fig. 9.
|
|
Complex formation in solution.
We previously demonstrated
direct binding in solution of HveC(346t) to a mutant form of gD from
HSV-1 KOS. We found that gD(290-299t) bound to HveC(346t) with a
stoichiometry of 2:1 and disrupted the putative HveC(346t) tetramer
(29). Here we analyzed the sizes of complexes formed between
gD(285t) and HveC(245t) by gel filtration (Fig. 8C and E). When
equimolar amounts of HveC(245t) and gD(285t) were mixed, a complex of
113 kDa eluted from the column, a size consistent with a complex
containing one dimer of gD and one dimer of HveC(245t) (51 plus 71 kDa), hence with a stoichiometry of 1:1. When twice the amount of
gD(285t) is added to HveC(245t) a peak of free gD(285t) dimer could be
detected on the profile (Fig. 8E) and by Western blot (data not shown). When initially present at equimolar concentrations, HveC(143t) and
gD(285t) formed a complex with an apparent size of 76 kDa (Fig. 8D), a
size consistent with a complex containing one dimer of each component
(29 plus 51 kDa), also with a stoichiometry of 1:1. No free receptor
was detected. When twice the amount of gD(285t) was initially present,
the peak shifted to 65 kDa (Fig. 8F). This is due to an excess of free
gD in fractions containing minimal amounts of HveC(143t), as detected
by Western blot analysis of the fractions (data not shown). Since the
1:1 ratio did not correlate with the stoichiometry obtained earlier
with gD(346t) and gD(290-299t), we repeated the study here with
HveC(346t) and gD(285t). In the presence of excess gD(285t), the
complex eluted with a size of 176 kDa (data not shown), a result
consistent with our previously observed complex made of two dimers of
gD and one dimer of HveC(346t), a stoichiometry of 2:1.
To confirm the stoichiometry of the gD(285t)-HveC(143t) complex, we
separated and quantified each component of the complex
by PAGE (Fig.
9). A fraction containing the complex
formed in
the presence of excess gD(285t) but separated from the peak
containing
free gD was analyzed at two different dilutions (Fig.
9,
lanes
5 and 6). By comparing the intensity of the silver-stained
proteins
in the fraction with known standards (lanes 1 to 4 and 7 to
10),
we determined that similar amounts of gD(285t) and HveC(143t)
were
present in the complex. This indicated a 1:1 ratio of HveC(143t)
to
gD(285t), supporting the ratio deduced from the size of the
complex.
This experiment could not be performed with the gD(285t)-HveC(245t)
complex because both protein monomers are too close in size to
be
sufficiently separated on a gel to allow accurate quantification.
When
the amounts of gD(285t) and HveC(346t) in the complex formed
in the
presence of excess gD(285t) were quantified by silver-stained
gel
analysis, the 2:1 ratio was obtained (data not shown), thus
confirming
our previous observations (
29).
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FIG. 9.
Quantitation of gD(285t) and HveC(143t) in the complex.
Two aliquots of a fraction containing the gD(285t)-HveC(143t) complex
separated by gel filtration (Fig. 8F) were loaded onto a 16%
polyacrylamide gel (lanes 5 and 6). Standards of known amounts of
HveC(143t) and gD(285t) were loaded in lanes 1 to 4 and lanes 7 to 10, respectively, with amounts of proteins as indicated under the gel.
After the silver staining of the gel, the intensity of the protein
bands was measured by densitometry. The amount of each protein in the
complex is indicated and is based on the comparison with the
standards.
|
|
| |
DISCUSSION |
When Ig-like molecules are used as cellular receptors by viruses,
the most membrane-distal domain is usually the site of direct contact
with the viral ligand (14, 16, 38, 40, 42). The HveC
V-domain fused to PVR Ig-domains 2 and 3 or directly anchored on the
cell membrane was shown to confer HSV susceptibility to normally
resistant cells (9). Cocchi et al. (9) also showed that the most distal Ig-domain of HveC was also the binding site
for HSV and for the neutralizing MAb R1.302. However, in that study,
the V-domain directly anchored to the cell membrane was poorly used as
a receptor, and the soluble construct consisting of the HveC V-domain
fused to the Ig Fc portion had a reduced capacity to bind gD. These
observations suggest that the second and/or third Ig domains are
necessary to ensure proper infectivity by influencing the availability
or the affinity to gD. In order to assess the contribution of each
Ig-like domain to gD binding, we produced soluble HveC truncations in
the baculovirus expression system. We generated three truncations of
HveC containing either the full ectodomain [HveC(346t)]
(29), the two distal domains [HveC(245t)], or the V-domain
alone [HveC(143t)] without the addition of substitutive Ig domains
such as the Ig Fc region. The ability of the three truncated forms of
HveC to bind virions and gD were compared by using several different
assays. We also examined the kinetics and stoichiometry of complex
formation with each form of HveC.
The HveC V-domain is sufficient for complete binding to gD.
The HveC V-domain was produced as a soluble 15-kDa glycoprotein and
appeared to be correctly folded, since it was recognized by the
conformation-dependent MAb R1.302. However, this shortest form of HveC
was altered by immobilization on an ELISA plate since it partially lost
its ability to bind MAb R1.302. This result is reminiscent of the
finding that the single PVR V-domain could not be detected by a
specific MAb after immobilization on an ELISA plate (2).
Also, stability of the ICAM-1 first Ig domain, where the binding site
for rhinovirus was mapped (38, 50), was influenced by
mutations in the second Ig domain; however, binding to rhinovirus was
not affected (7, 50).
Although the immobilized HveC V-domain showed reduced binding to gD, it
could efficiently compete with HveC(346t) for binding
with gD in
solution. Furthermore, the single V-domain bound HSV
particles and
blocked infection of HveC expressing CHO cells and
neuroblastoma cell
lines as well as the full ectodomain. This
finding confirms previous
observations with the HveC V-domain
fused to the Ig Fc region
(
9) and indicates that additional
Ig-like domains are
dispensable at least for the binding of gD.
In the case of MHV receptor
(bgp1
a) (
57), the binding of viral spike protein
to the Ig-like domain
1 is influenced by adjacent Ig-like domains
(
14). In this instance,
the first two Ig-domains of soluble
bgp1
a have lower affinity for MHV and reduced neutralizing
activity.
However, when Ig-domain 4 was placed in second position, it
affected
binding and neutralizing activities differently than the
normal
Ig-domain 2 (
61).
Affinity of the HveC ectodomain for gD.
To study the HveC-gD
interaction in real time, we used biosensor technology. First, kinetic
and affinity values were calculated for the interaction between the
full HveC ectodomain and three truncated forms of HSV-1 gD. The
affinity of HveC(346t) for gD(306t) from the KOS strain of HSV-1, which
we consider the wild-type form, was in the micromolar range, with a
KD comparable to that observed for the
interaction between gD(306t) and the structurally unrelated receptor,
HveA (47, 59). By ELISA, the C-terminal truncation gD(285t),
which lacks functional region IV, and a gD(rid1t) protein, with a point
mutation at position 27, showed an enhanced binding to HveC(346t)
(29, 47, 59). Biosensor analysis confirmed an 80-fold
enhancement of affinity of gD(285t) for HveC and an enhancement of
about 20 times of the overall affinity of gD(rid1t) for HveC. In each
case the variation in the affinity of these gD proteins for HveC was
caused by changes in the rapidity of complex formation (on rates),
whereas the off rates were similar. This suggests that, once the
complex is formed, its stability is independent of the gD form bound.
This observation also held true in the case of HveA binding to most gD
mutants studied (47, 59).
It is of note that, unlike the interaction between gD and HveA, the
complex formation between gD and HveC did not reach equilibrium
even
after prolonged contact time, regardless of which form of
soluble gD or
HveC was used. In addition, when we plotted the
concentration-dependent
on rate (
kobs) versus concentration we
obtained
a convex curve, indicating that the ligand immobilized
on the chip was
heterogeneous (data not shown). HveC heterogeneity
might result from
its direct immobilization on the biosensor chip
(
5).
However, the potential for self-association of HveC could
be
responsible for heterogeneity and could lead to a more complicated
interaction with gD than the simple 1:1 interaction model used
to fit
biosensor
data.
The HveC V-domain affinity for gD compares to the full HveC
ectodomain.
We compared the affinity of gD for the HveC V-domain
alone with the affinity of gD for the complete HveC ectodomain. In the case of gD(306t), the affinity for HveC(143t) was slightly higher than
for HveC(346t), whereas no differences were observed in the affinity of
gD(285t) or gD(rid1t) for the two receptor forms. This indicates that
the V-domain alone retains all the necessary elements to allow binding
to gD variants spanning a broad range of affinity. A biosensor approach
was used to study ICAM-1 binding to rhinovirus (7).
Truncated ICAM-1 proteins containing the first two domains could be
generated without significantly affecting the affinity for rhinovirus.
However, an ICAM-1 protein truncated five amino acids upstream, at the
bottom of domain 2, had an increased off rate correlated with a
decreased ability to block infection and to bind rhinovirus (7,
20, 36). On the contrary, the HveC V-domain alone did not display
any variation in the rate of complex dissociation.
Oligomerization of HveC in solution and complex formation with
soluble gD.
When run on a size exclusion chromatography column,
HveC(346t) had a molecular size of 176 kDa, a result consistent with
the formation of a tetramer in solution (29). In contrast,
the sizes of HveC(143t) and HveC(245t) in solution were consistent with formation of dimers. By analogy, ICAM-1 domains 1 and 2 also form dimers via the interaction of two domains 1 (3, 8). Our data
strongly suggest that the third Ig-like domain (closest to the
transmembrane domain) is involved in a higher order of oligomerization of HveC (i.e., a tetramer). In a model proposed by Casasnovas et al.
(8) for ICAM-1, the Ig-like domain 5 close to the
transmembrane domain is also proposed to be involved in
oligomerization. We analyzed the complexes formed in solution between
each truncated form of HveC and gD(285t). In the case of HveC(143t) and
HveC(245t), the data suggested a complex consisting of one gD dimer and
one HveC dimer. The 1:1 ratio obtained in both complexes is in contrast to the 2:1 ratio of gD(290-299t) (29) or gD(285t) bound
to HveC(346t). It is possible that the higher degree of oligomerization of HveC(346t) in solution influences the stoichiometry of the complex
with gD. It should be noted that in spite of the apparent difference in
stoichiometry, gD(285t) binds to HveC(143t) and to HveC(346t) with
equal affinity. In addition, all of the biosensor data fit the 1:1
model, which suggests that there is not a second gD binding site on
HveC Ig domain 2 or 3. Since the size of the gD-HveC(346t) complex is
similar to that of HveC(346t) alone, it is likely that association with
gD may disrupt the HveC tetramer into dimers. This distinct property of
HveC(346t) suggests that the V-domain is somehow involved with domain 3 in tetramer formation. Further studies need to be done to better
correlate the oligomerization of HveC and stoichiometry of the gD-HveC
complex. The cell membrane anchored HveC V-domain alone was markedly
less efficient as a receptor for HSV entry (9). Since all of
the elements necessary for binding gD with full affinity are present in
the V-domain of HveC, the decrease in efficiency of the V-domain alone
might be due to poor availability, structural instability or incorrect oligomerization due to absence of Ig-domains 2 and/or 3. These observations might become more relevant once the oligomeric status of
HveC at the surface of cells is determined.
| |
ACKNOWLEDGMENTS |
This investigation was supported by Public Health Service grants
AI-18289 to R.J.E. and G.H.C. and AI-36293 to P.G.S. from the National
Institute of Allergy and Infectious Diseases (NIAID), grant NS-30606 to
R.J.E. and G.H.C. from the National Institute of Neurological Diseases
and Stroke, and grant CA-21776 to P. G. S. from the National
Cancer Institute. C.K. was supported by a fellowship (823A-053464) of
the Swiss National Science Foundation. R.J.G. was supported by a
fellowship from the NIAID (F32 AI-09471). We thank the Schools of
Dental and Veterinary Medicine of the University of Pennsylvania for
supplying funds for the purchase of the Biacore X.
We thank William T. Moore and John D. Lambris of the Protein Chemistry
Laboratory of the School of Medicine of the University of Pennsylvania,
supported by core grants of the Diabetes and Cancer Centers (DK-19525
and CA-16520), for mass spectrometric analysis. We thank Ruliang Xu for
the M3A cells and S. McClellan (Beckman/Coulter) for R1.302 MAb. We are
grateful to Nicholas Fasano for technical assistance and to Sharon
Willis for critical reading of the manuscript and helpful discussion.
| |
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:
krumm{at}biochem.dental.upenn.edu.
| |
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Abstract
Introduction
