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Journal of Virology, July 1999, p. 5681-5687, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Inhibition of Herpes Simplex Virus gD and
Lymphotoxin-
Binding to HveA by Peptide Antagonists
Maria Rosa
Sarrias,1
J. Charles
Whitbeck,2,3
Isabelle
Rooney,4
Lynn
Spruce,1
Brian K.
Kay,5
Rebecca I.
Montgomery,6
Patricia G.
Spear,6
Carl F.
Ware,4
Roselyn J.
Eisenberg,2
Gary H.
Cohen,2,3 and
John D.
Lambris1,*
Laboratory of Protein Chemistry, Department of Pathology and
Laboratory Medicine, School of
Medicine,1 Department of Microbiology,
School of Veterinary Medicine,2 and
Center for Oral Health Research, School of Dental
Medicine,3 University of Pennsylvania,
Philadelphia, Pennsylvania 19104; Division of Molecular
Immunology, La Jolla Institute for Allergy and Immunology, San
Diego, California 921214; Department of
Pharmacology, University of Wisconsin, Madison, Wisconsin
537065; and Department of
Microbiology-Immunology, Northwestern Medical School, Chicago,
Illinois 606116
Received 27 January 1999/Accepted 29 March 1999
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ABSTRACT |
The herpesvirus entry mediator A (HveA) is a recently characterized
member of the tumor necrosis factor receptor family that mediates the
entry of most herpes simplex virus type 1 (HSV-1) strains into
mammalian cells. Studies on the interaction of HSV-1 with HveA have
shown that of all the viral proteins involved in uptake, only gD has
been shown to bind directly to HveA, and this binding mediates viral
entry into cells. In addition to gD binding to HveA, the latter has
been shown to interact with proteins of tumor necrosis factor
receptor-associated factor family, lymphotoxin-
(LT-), and a
membrane-associated protein referred to as LIGHT. To study the
relationship between HveA, its natural ligands, and the viral proteins
involved in HSV entry into cells, we have screened two phage-displayed
combinatorial peptide libraries for peptide ligands of a recombinant
form of HveA. Affinity selection experiments yielded two peptide
ligands, BP-1 and BP-2, which could block the interaction between gD
and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO
cells transfected with an HveA-expressing plasmid. When we analyzed
these peptides for the ability to interfere with HveA binding to its
natural ligand LT-, we found that BP-1 inhibited the interaction of
cellular LT- with HveA. Thus, we have dissected the sites of
interaction between the cell receptor, its natural ligand LT- and
gD, the virus-specific protein involved in HSV entry into cells.
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INTRODUCTION |
The herpesvirus entry mediator A
(HveA; formerly named HVEM) is a member of the tumor necrosis factor
receptor (TNFR) superfamily and has been shown to act as a receptor for
herpes simplex virus (HSV) (16). Expressed in otherwise
nonpermissive CHO cells, it rendered these cells susceptible to entry
by several HSV strains. This binding was inhibited by recombinant
soluble HveA and antibodies to HveA. In addition to the involvement of
HveA in the entry of extracellular virus, it was found that it
participates in cell-to-cell transmission of the virus (22,
30). The HSV protein mediating its binding with HveA has been
shown to be the glycoprotein D (gD), as it binds directly to a
soluble form of HveA [HveA(200t)] (34) in a specific
and saturable manner and inhibits the binding of HSV to HveA-expressing
cells (20, 21, 27-29, 34).
In addition to its involvement in HSV entry, several findings suggest
that HveA plays a role in the activation of the host immune response.
For example, HveA, predominantly expressed in lymphocyte-rich tissues,
has been shown to interact with several members of the TNFR-associated
factor (TRAF) family of proteins. This interaction leads to the
activation of transcriptional regulators such as NF-
B, Jun
N-terminal kinase, and AP-1 (8, 14). There are two known
ligands for the extracellular domain of HveA, lymphotoxin- (LT-)
and the membrane-associated protein referred to as LIGHT. LIGHT is a
newly identified lymphotoxin homolog which is expressed by T cells upon
induction with phorbol myristate acetate and Ca2+ ionophore
and competes with a soluble form of HSV gD (gDt) for binding to HveA.
Thus, either LT- or LIGHT may modulate HSV infection by competing
for HveA binding and vice versa, which has led to the hypothesis that
gD may modify HveA-signaling activities during entry or egress of HSV,
thus modulating the immune response of the host (15).
The mode of HveA interaction with its ligands, as well as whether
HveA interacts with them via multiple sites or whether these ligands share binding sites, is not known. The rich but uncharted molecular diversity that is offered by the surface of the HveA molecule
calls for an equally diverse approach to searching for ligands that are
complementary and specifically interactive with particular sites.
Within the last 10 years, random peptide libraries have provided a rich
source of structural diversity (10). They have proved to be
a useful tool in identifying the peptide epitopes recognized by
particular monoclonal antibodies as well as mimetics of ligands for
various proteins.
In this study, our goal was to study the interaction between HveA, its
natural ligands, and HSV gD. To this end, we have used recombinant HveA
to screen two phage-displayed combinatorial peptide libraries and have
selected two peptide ligands that differentially inhibit binding of gDt
and LT- to the receptor. Furthermore, one of these peptides was able
to block HSV entry into HveA-expressing CHO cells.
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MATERIALS AND METHODS |
Chemicals and buffers.
All chemicals and reagents used for
peptide synthesis were purchased from Applied Biosystems (Foster City,
Calif.) with the exception of the F-moc (9-fluorenylmethoxycarbonyl)
amino acids, which were obtained from Nova Biochem (San Diego, Calif.).
Protein expression.
The production and purification of
HveA(200t), gD-1(306t), gD-1(
290-299t), and LT- from recombinant
baculovirus-infected cells have been described elsewhere (5, 19,
21, 25, 31, 34, 35). Briefly, the cDNA of interest was amplified
by PCR for each protein with five or six C-terminal His codons, and a stop codon was added to the downstream primer. The run of His codons
was added to provide a binding site for a nickel-nitrilotriacetic acid-agarose resin used in the purification of the expressed protein. The PCR-amplified products were cloned into the pVT-Bac vector (31), with the honeybee mellitin signal sequence replacing
their own signal sequences.
The resulting constructs were recombined into baculovirus
(Autographa californica nuclear polyhedrosis virus) by
cotransfection with Baculogold DNA (Pharmingen, San Diego, Calif.).
Spodoptera frugiperda Sf9 (GIBCO BRL) cells grown in
suspension cultures were infected with one of the recombinant
baculoviruses at a multiplicity of infection of 4. At 48 to 72 h
after infection, the supernatants were cleared by centrifugation,
concentrated, and then dialyzed against phosphate-buffered saline
(PBS). The proteins were affinity purified over a column of
nickel-nitrilotriacetic acid-agar resin and eluted with increasing
concentrations of imidazole (0.01 to 0.25 M) in 0.02 M phosphate buffer
(pH 7.5)-0.5 M NaCl. The eluates were dialyzed against PBS and concentrated.
Antibodies.
A polyclonal antibody (R140) against HveA was
generated by immunizing a rabbit with baculovirus-produced HveA (200t)
as previously described (30, 34). A polyclonal antibody
specific for gD (R7) was raised against gD-2 isolated from
virus-infected cells as previously described (9). The
production of a monoclonal antibody to LT- (AG9) has been described
elsewhere (4).
Cells and viruses.
CHO cells were grown in Ham's F-12
medium supplemented with 10% fetal calf serum (FCS). CHO-K1 cells
expressing HveA (CHO-HveA cells) and CHO-K1 cells expressing HveC
(CHO-HveC cells) were grown in Ham's F-12 medium supplemented with
10% FCS and 200 µg of G418 per ml. KOS-hrR3 virus is a
mutant KOS virus in which the Escherichia coli lacZ gene is
positioned in the ribonucleotide reductase large subunit locus (ICP6)
and is under the transcriptional control of the ICP6 promoter. This
virus was propagated in African green monkey kidney (Vero) cells
expressing the large subunit of HSV type 1 ribonucleotide reductase
(7). Vero cells were grown in Dulbecco's minimal essential
medium, supplemented with 5% FCS, at 37°C.
Construction and screening of the phage libraries.
The
27-mer library consisted of 2 × 108
recombinants, each expressing the peptide sequence
SRX12(S/P/T/A)A(V/A/D/E/G)X12SR at
the N terminus of mature pIII of bacteriophage M13. The library was
constructed by annealing and extending two long degenerate oligonucleotides, complementary to each other at their 3' termini (11, 23). The six nucleotides of complementarity form a
SacII restriction site and encode the tripeptide
(S/P/T/A)A(V/A/D/E/G) (1). The 12-mer library consisted of
108 recombinants, each expressing the peptide sequence
X12 at the N terminus of mature pIII. The library was
constructed by annealing and extending two oligonucleotides, one of
which was degenerate.
The coding scheme of the random amino acids in both libraries was NNK,
where N represents equimolar ratios of A, C, G, or
T and K represents G
or T. In this coding strategy, the 20 amino
acids are encoded by 32 codons, each with certain amino acids
occurring once (C, D, E, F, H, I,
K, M, N, Q, W, Y), twice (A,
G, P, V, T), or three times (L, R,
S).
HveA-binding phage were isolated by screening the 27-mer and 12-mer
libraries as previously described (
2,
11). Microtiter
wells
(Nunc, Inc., Naperville, Ill.) were coated overnight at
4°C or for
2 h at 22°C with 500 ng of HveA(200t) and blocked with
PBS
containing 1% bovine serum albumin (BSA) for 1 h at 22°C.
After
washing, 6 × 10
11 PFU of each library was added to
each well and incubated for
1 h at 22°C. The wells were washed
two times with PBS containing
0.05% Tween 20. Bound phage particles
were eluted with 100 mM
glycine-HCl (pH 2.3), and the samples were
immediately neutralized
with 200 mM sodium phosphate (pH 7.4). The
eluted phage particles
were amplified in
E. coli DH5
F',
and the affinity selection
procedure repeated twice more. The amplified
phage mixture obtained
after the third round of amplification was
plated, and positive
phages were identified by confirming their ability
to bind to
HveA(200t) in an enzyme-linked immunosorbent assay (ELISA)
in
which bound phages were detected by peroxidase-labeled anti-M13
antibody (Amersham Pharmacia Biotech, Piscataway, N.J.). DNA was
prepared from positive phage stocks and subjected to dideoxy
sequencing
as previously described (
24).
Synthesis and purification of peptides.
All peptides were
synthesized on an Applied Biosystems peptide synthesizer (model 431A),
using F-moc amide resin. The side chain protecting groups were
Cys(Trt), Cys(Acm), Arg(Pmc), Ser(tBu), and Tyr(tBu).
The peptides cyclic BP-2 and scrambled BP-2 were cyclized on resin by
treatment with 1.5 equivalents of thallium III trifluoroacetate
in
dimethylformamide for 3 h at 22°C. The peptide-resin beads
were
then washed with dimethylformamide, methanol, and
methanol-dichloromethane
(60:40) and dried under vacuum. The peptides
were cleaved from
the peptide-resin by treatment with 87.5%
trifluoroacetic acid
(TFA), 5% phenol, 5% water, and 2.5%
triisopropylsilane for 3
h at 22°C, harvested from the reaction
mixture by filtration,
and precipitated in cold ether. The peptide
precipitates were
extracted three times with cold ether, the pellets
were dissolved
in 50% acetonitrile containing 0.1% TFA, and the
samples were
lyophilized.
Disulfide oxidation of BP-1 was performed after cleavage from the resin
by stirring a 0.15 mM solution of peptide in 0.1 M
ammonium bicarbonate
(pH 8.0, and bubbling with oxygen at 22°C
for 48 h. Linear BP-1
was obtained by maintaining the side chain
protecting groups on the Cys
residues Cys(Acm). All of the crude
peptides were dissolved in 10%
acetonitrile containing 0.1% TFA
and purified by reversed-phase
high-performance liquid chromatography
on an automated system (Prep-LC
4000; Waters, Milford, Mass.)
with a C
18 column (Vydac,
Western Analytical Products, Inc., Murrieta,
Calif.). The column was
initially equilibrated with 5% buffer
B (0.1% TFA in water) for 15 min at a flow rate of 20 ml/min,
and peptide fractions were eluted with
a 50-min linear gradient
of 1 liter of 5 to 90% buffer B (0.1% TFA in
acetonitrile) at
a flow rate of 20 ml/min. The elution profile of the
peptide fractions
was monitored by UV detection at 230 nm, and the
major peak containing
the desired peptide was collected and
lyophilized. The purity
of the final products was assessed by
analytical high-performance
liquid chromatography and matrix-assisted
laser desorption mass
spectrometry, using a time-of-flight mass
spectrometer (MicroMass
TofSpec; Micromass Inc., Beverly, Mass.)
(
3,
17,
18).
ELISAs.
Several ELISAs were performed to analyze the
interaction between HveA, the isolated phage peptides, gD, and LT-.
In these assays, microtiter wells were coated for 2 h at 22°C
with 40 ng of HveA(200t), human factor H, trout complement C3, BSA,
milk, or ovalbumin. Nonspecific binding in the wells was blocked with PBS containing 1% BSA for 1 h at 22°C. For competition assays, serial dilutions of gD-1(306t), gD-1(290-299), BSA, peptide BP-1 or
BP-2, or an unrelated cyclic peptide (ICVVQDWGHHRCT) was added to each
well and incubated for 30 min at 22°C. Recombinant protein [gD-1(306t) at 0.4 µg/ml, gD-1(290-299t) at 0.1 µg/ml, or
LT- at 20 nM] or phage supernatant was added to each well, and the samples were incubated for 1 h. The wells were washed twice with PBS containing 0.05% Tween 20 and incubated with either (i) a 1:1,000
dilution of a peroxidase-labeled anti-M13 antibody, (ii) a 1:400
dilution of an anti-gD polyclonal antibody (R7), or (iii) an
anti-LT- monoclonal antibody (AG9; 1 µg/ml) for 1 h at
22°C. The wells were washed with PBS containing 0.05% Tween 20 and
then incubated with a 1:1,000 dilution of peroxidase-conjugated goat anti-rabbit immunoglobulin G (for polyclonal antibody detection) or
goat anti-mouse immunoglobulin G (for monoclonal antibody detection) (Bio-Rad Laboratories, Richmond, Calif.). The plates were incubated for
an additional 30 min at 22°C. Color was developed by adding 2.2'-azino-di-[3-ethylbenzthiazolinesulfonate (6)] (ABTS; Boehringer Mannheim) and 0.05% hydrogen peroxide, and the optical density was
read at 405 nm. Net gD-1 or LT- binding was calculated by subtracting the readings of secondary antibody to HveA binding from the
readings of ligand binding to HveA.
HSV entry assays.
CHO-HveA, CHO-HveC, or Vero cells, were
plated in 96-well dishes and incubated overnight. The cells were
chilled at 4°C for 10 min, and the medium was replaced with Ham's
F-12 (CHO-HveA and CHO-HveC cells) or Dulbecco's modified Eagle medium
DMEM (Vero cells) with 10% fetal bovine serum and 10 mM HEPES,
containing various concentrations of peptide, previously filtered
through a 0.2-µm-pore-size filter (Corning Glass Works, Corning,
N.Y.). The plates were rocked at 4°C for 90 min, at which time
KOS-hrR3 virus (5 × 105 PFU/well) was
added. The plates were rocked at 4°C for an additional 90 min and
then incubated at 37°C for 5 h. Cells were lysed with 20%
Nonidet P-40, and substrate
(o-nitrophenyl-
-D-glucopyranoside) was added
to each well. -Galactosidase activity (milli-optical density units
per minute) was measured at various time points with a Spectromax 250 ELISA reader at 560 nm.
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RESULTS |
Isolation and characterization of HveA-binding phages.
To
isolate peptide ligands to the extracellular domain of HveA, we
screened two phage libraries displaying combinatorial peptides 27 and
12 amino acids in length. Phage particles expressing HveA-binding peptides were affinity selected against HveA(200t) immobilized on
microtiter plate wells. After three rounds of selection, individual phage clones were isolated and tested for binding to HveA.
The binding of the phages was deemed to be specific, as they did not
bind to trout complement C3, milk, ovalbumin, BSA, or
human factor H
(Fig.
1B). In addition, soluble
gD-1(306t) could
compete the binding of the phage to immobilized
HveA(200t) (Fig.
1A).
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FIG. 1.
Specific binding to HveA of positive clones isolated
from two phage-displayed random peptide library. (A) Microtiter wells
were coated with HveA(200t) and incubated with dilutions of gD-1(306t)
or BSA. Phage supernatant from five positive clones isolated from the
26-mer library was added. Similar results were obtained with positive
clones from the 12-mer library. (B) Microtiter wells were coated with
HveA(200t), BSA, milk, trout complement C3, human complement factor H,
or ovalbumin (OVA). Phage supernatant from a positive clone isolated
from a 12-mer library was added. Bound phage was detected with
peroxidase-conjugated anti-M13 antibody and ABTS peroxidase
substrate.
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DNA obtained from several positive clones was purified and sequenced.
All positive clones from each of the libraries had the
same sequence,
indicating that the clones had been amplified during
the second and
third rounds of biopanning. The deduced amino acid
sequences of the
clones were SISCSRGLVCLLPRLTNESGNDRFDS (BP-1)
from the 27-mer
library and GSCDGFRVCYMH (BP-2) from the 12-mer
library. The
amino acid sequence of the positive clones from the
27-mer library does
not agree with the original design of the
library,
X
12(S/P/T/A)A(V/A/D/E/G)X
12. The central
sequence of
the peptide in this region was PR instead of the expected
(S/P/T/A)A(V/A/D/E/G).
Thus, BP-1 is only 26 amino acids
in length. We have searched
the GenBank (DNA) and SwissProt
(protein) databases for sequences
showing similarity to BP-1 and BP-2,
using BlastP, BlastN, and
BlastX, and found
none.
To further characterize the binding properties of the isolated clones,
we synthesized two peptides corresponding to the deduced
amino acid
sequences of the phage-displayed peptides (Fig.
2).
It is interesting that although the
two isolated clones are not
similar in sequence, binding of phage
displaying BP-1 to HveA
was inhibited in an ELISA by peptide BP-2, and
vice versa (Fig.
3).
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FIG. 3.
Inhibition of phage binding to HveA by BP-1 and BP-2.
Microtiter wells were coated with HveA(200t) and incubated with
decreasing concentrations of peptide and phage displaying BP-2 (A) or
BP-1 (B). Bound phage particles were detected with
peroxidase-conjugated anti-M13 antibody and ABTS peroxidase
substrate.
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Inhibition of gD-1 binding to HveA.
The two synthetic
peptides, BP-1 and BP-2, were next tested for the ability to compete
the binding of gD-1 to the HveA receptor (Fig.
4). Binding of soluble wild-type gD-1,
gD-1(306t), to immobilized HveA(200t) was reduced by BP-2 to a level of
80% and by BP-1 to a level of 20% at a concentration of 0.5 mM (Fig.
4A). Binding of the mutant form gD-1(290-299t), which has a
100-fold-higher affinity for HveA, was inhibited to a level of 40% by
BP-2 (Fig. 4B). Conversely, no inhibitory effect was observed with a
blocked cysteine [BP-1 (4,10 Acm)], alanine replacement [BP-2 (3,9 Ala)], a peptide with a scrambled sequence of BP-2, or a cyclic
peptide with a completely different sequence (Fig. 4C). Since these
negative results included peptides BP-1 (4,10 Acm) and BP-2 (3,9 Ala), in which no disulfide bond is present, it appears that disulfide bond
formation is important for maintaining the inhibitory activity of both
BP-1 and BP-2.
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FIG. 4.
Inhibition of gD-1(306t) binding to HveA by BP-2 and
BP-1. Microtiter wells were coated with HveA(200t) and incubated with
decreasing concentrations of BP-2 or BP-1. gD-1(306t) (A) or
gD-1(290-299t) (B) was added. (C) In this ELISA, HveA-coated plates
were incubated with decreasing concentrations of BP-2 and control
peptides before gD-1(306t) was added. Bound gD-1 was detected with a
polyclonal antibody anti-gD (R7) followed by incubation with
peroxidase-conjugated secondary antibody and ABTS. The data were
obtained by subtracting nonspecific binding of anti-gD antibody to
peptide bound to HveA.
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Inhibition of LT- binding to HveA.
The effect of synthetic
peptides BP-1 and BP-2 on LT- binding to HveAt was analyzed by
ELISA. HveAt was coated onto the wells of a microtiter plate and
incubated with various concentrations of peptides along with 20 nM
LT-, and the amount of LT- bound was detected with a monoclonal
antibody. As seen in Fig. 5, binding of
soluble LT- to HveAt was inhibited by BP-1, whereas neither BP-2 nor
the control peptides had an inhibitory effect. This assay indicates
again that peptide BP-1 requires cyclization to bind to HveAt. On the
other hand, BP-2 did not inhibit LT- binding to the receptor,
suggesting that BP-1 and BP-2 bind to different sites on HveAt.
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FIG. 5.
Inhibition of LT- binding to HveA by BP-1, BP-2, and
control peptides. HveA-coated microtiter plate wells were incubated
with concentrations of peptides, and LT- was added. Bound LT- was
detected with an anti-LT- monoclonal antibody (AG9), followed by
incubation with peroxidase-conjugated secondary antibody and ABTS
peroxidase substrate.
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Inability of gD to inhibit LT- binding to HveA.
Given that
we had discovered that the peptides appeared to bind two different
sites on HveA, we investigated whether gD and LT- bind HveA in a
competitive manner (Fig. 6). An ELISA was designed to compare the bindings of gDt and LT- to HveAt. In this
assay, HveAt was first immobilized on the plate and incubated with a
constant amount of LT- in the presence of various amounts of gDt.
The amount of LT- bound to HveAt was detected with a monoclonal
antibody to LT- (AG9). To confirm that gDt remained bound to HveAt
during the wash steps, bound gDt was detected with a polyclonal anti-gD
antibody, R7, in separate wells of the same ELISA plate. We found that
the same amount of LT- bound to HveAt regardless of how much gDt was
added to the plate wells. Thus, gDt had no inhibitory effect on LT-
binding to the receptor, suggesting that these proteins do not compete
for the same binding sites on HveAt.
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FIG. 6.
Lack of competition by gD on LT- binding to HveA.
HveA-coated microtiter plate wells were incubated with various
concentrations of gD, and LT- was added. Bound LT- and gD were
detected with an anti-LT- monoclonal antibody (AG9) and a polyclonal
antibody (R7), respectively, followed by incubation with a
peroxidase-conjugated secondary antibody and ABTS peroxidase
substrate.
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Inhibition of HSV-1 entry into cells by BP-1 and BP-2.
The
inhibition of gD-1 binding to HveAt by BP-2 raised the question of
whether this peptide has any effect on viral entry into mammalian
cells. To test this possibility, we examined the effect of the 12-mer
peptide on HSV-1 entry into CHO-HveA, CHO-HveC, and Vero cells. Cell
monolayers were incubated with peptide at 4°C for 90 min and then
infected with the -galactosidase reporter virus KOS-hrR3.
BP-2 blocked HSV entry into CHO-HveA cells; in contrast, neither BP-1
nor any of the control peptides blocked virus entry (Fig.
7A). Neither peptide had any effect on
HSV entry into Vero (Fig. 7B) and CHO-HveC cells (data not shown)
cells. Thus, although both BP-1 and BP-2 bind to HveA, only BP-2 is
able to inhibit gD-1 binding to HveA effectively enough to block viral entry into CHO-HveA cells. This result is consistent with the data in
Fig. 4 showing that only BP-2 effectively blocked the binding of gDt to
HveAt.
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FIG. 7.
BP-2 can block HSV entry into CHO-HveA cells (A) but not
into Vero cells (B). Cell monolayers were incubated with dilutions of
peptides at 4°C for 90 min. KOS-hrR3 was then added and
incubated for an additional 90 min at 4°C for adsorption. Five hours
later (at 37°C), the cells were lysed and -galactosidase was
measured. The data are the averages of duplicate wells; background
-galactosidase activity (cells alone) has been subtracted.
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DISCUSSION |
Several receptors of HSV that mediate postattachment entry into
the cell have recently been described. The first to be characterized was HveA, a member of the TNFR family, which allows entry of most HSV
strains into nonpermissive CHO cells (16). This process was
shown to be mediated by direct binding of the viral glycoprotein gD to
HveA (19, 34). In addition to binding to gD, the same cell
surface protein was found to interact with several host proteins including, LT-, LIGHT, and TRAF proteins (8, 14, 15).
In this study, we used two different phage-displayed combinatorial
peptide libraries to identify HveA-binding peptides that affect the
interaction of the receptor with its ligands. We have dissected the
interaction between the proteins gD, LT-, and HveA in the context of
viral entry.
After three rounds of biopanning, several positive clones from each
library that bound specifically to HveA(200t) were isolated. These
clones did not bind to any of several other proteins, including trout
complement C3, human complement factor H, ovalbumin, and milk. This
specificity was corroborated by the fact that soluble gD-1(306t)
inhibited phage binding to HveAt. To our surprise, all of the positive
clones from each library had the same sequence. This result suggests
that the unique HveA-binding clones were amplified during the second
and third rounds of library biopanning. It is interesting that the two
identified peptide ligands, while differing in primary structure, each
had two cysteine residues. Experiments with Ala-substituted Cys or side
chain-blocked Cys demonstrated that the intramolecular disulfide bond
is necessary for peptide binding to HveA. It was found that both
synthetic peptides BP-1 and BP-2 were unable to inhibit gDt binding to
HveAt when their two Cys residues were not disulfide linked. The same effect was observed for BP-1 in relation to inhibition of LT- binding to HveAt; the linear form of this peptide was unable to inhibit
the LT--HveA interaction. These results suggest that the
intramolecular disulfide bond is essential to maintain the binding and
inhibitory activities of these peptides. The binding of phage clones to
HveA suggests that the disulfide bond in the peptides is also formed
when they are expressed on the surface of the phages; the oxidizing
environment of the bacterial periplasm has been shown to allow
disulfide bond formation between Cys residues in phage-displayed
peptides (12).
The marginal effect observed on the inhibition of fluid-phase gDt
binding to HveAt by BP-1 contrasts that seen for BP-2, which inhibited
not only the binding of soluble gD-1(306t) but also the binding of
soluble gD-1(290-299t), a mutant form of gD having a 100-fold-higher
affinity for HveAt (21, 34, 36). Furthermore, BP-2 blocked
viral entry into CHO-HveA cells, where gD-1(306t) had failed to do so
in previous studies. It is possible that BP-2 has a higher affinity for
HveA than does the wild-type gD-1(306t), and therefore large
amounts of gD-1(306t) are needed for inhibition. The failure of
BP-2 to block viral entry into CHO-HveC cells suggests that this
peptide binds specifically to HveA. Since BP-2 also fails to block HSV
entry into Vero cells, it appears likely that entry mediators other
than or in addition to HveA are expressed. Alternatively, it is
possible that the site in HveA to which the peptides bind is not
conserved in the primate Vero cell homolog of HveA.
The differential inhibitory effects of the two peptides on LT- and
gDt binding to HveAt suggest that BP-1 and BP-2 bind to different sites
on HveA. Because binding of BP-1 to HveAt is inhibited by BP-2, and
vice versa, we suggest that either the peptides bind to overlapping
sites on HveAt or the binding of one peptide to HveAt induces
conformational changes in the receptor that influence the binding of
the other peptide (26) (Fig.
8). In the first model, BP-1 and BP-2 may
partially share binding sites on HveAt and thus compete for binding to
those sites. Accordingly, although LT- binding to HveAt was
inhibited only by BP-1, the HveA site to which gD binds could include
structural elements involved in the binding of both peptides. In this
way, both peptides could affect the interaction between gDt and HveAt.
However, BP-1 inhibits only partially the binding of gD to HveA, and
thus it appears that the gD-binding site on HveA is nearer the BP-2
site than the BP-1 site. The remarkable inhibitory effect of BP-2 on
gDt binding to HveAt, which led to inhibition of HSV entry into
CHO-HveA cells, suggests that BP-2 may have a high affinity for the
receptor. In addition, BP-2 may compete with gDt for more than one
binding site on HveAt. This possibility is consistent with a recent
study in which monoclonal antibodies recognizing two different regions on gD, Ib (amino acids 222 to 252) and VII (amino acids 11 to 19), were
found to block HSV binding to HveA (19). If the peptides bound to overlapping sites on HveA, then gD and LT- would bind to
adjacent sites on the receptor.
A second interpretation of our data is that binding of the peptides to
the receptor causes conformational changes in HveA. Thus, BP-1 binding
to HveAt could cause a conformational change in the receptor that
interferes with BP-2, LT-, and gDt binding to HveA. On the other
hand, BP-2 binding to HveAt could alter BP-1 and gDt binding sites on HveA.
HveA is not the only mediator of HSV entry into the cell. Other cell
surface receptors have recently been isolated and are currently being
studied (6, 33); the role and importance of these new
receptors in HSV entry have yet to be determined. Viral entry is a key
step in infection, and we have now succeeded in isolating a peptide
that blocks the interaction between HveA and HSV in vitro. Furthermore,
we have identified one site on HveA, where BP-2 binds, which when it is
occupied prevents viral uptake but not the binding of a cellular
ligand, LT-. Therefore, screening chemical compound libraries for
those that displace BP-2 but not BP-1 could lead to the development of
a useful drug to prevent HSV infection. We conclude that although
further studies are needed to assess the potential therapeutic
usefulness of BP-2, we believe that peptides BP-1 and BP-2 are
promising tools for analyzing the mechanisms of viral entry into cells
and HveA-host ligand interactions and functions.
| |
ACKNOWLEDGMENTS |
We thank A. Sahu, W. T. Moore, and A. Soulika for helpful
suggestions, D. McClellan for editorial assistance, and Yvonne
Harrison-Shahan for excellent technical assistance.
This work was supported by National Institutes of Health grants
NS-36731 and NS-30606, National Institute of Allergy and Infectious Diseases grants AI-30040 and AI-18289, and Cancer and Diabetes Centers
Core Support grants CA-16520 and DK-19525.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Protein
Chemistry Laboratory, Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Philadelphia, PA 19104-6079. Phone: (215)
662-6165. Fax: (215) 573-2059. E-mail:
lambris{at}mail.med.upenn.edu.
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Journal of Virology, July 1999, p. 5681-5687, Vol. 73, No. 7
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
