(Received for publication, March 6, 1997, and in revised form, May 28, 1997)
From the Echovirus 22 (EV22) is a picornavirus forming a
distinct molecular cluster together with echovirus 23. EV22 has an
Arg-Gly-Asp (RGD) peptide motif in its capsid protein VP1; similar
motifs are known to mediate many cell-cell and microbe-host
interactions. To identify peptide sequences that specifically bind to
EV22 and potentially play a role in receptor recognition, we have used here peptide libraries displayed in filamentous phage. We isolated an
EV22-binding motif CLRSG(R/F)GC. The synthetic CLRSGRGC peptide was
able to inhibit EV22 infection. The infection was also inhibited by an
RGD-containing peptide representing the C terminus of the EV22 capsid
protein VP1 and CWDDGWLC (an RGD-binding peptide; Pasqualini, R.,
Koivunen, E., and Ruoslahti, E. (1995) J. Cell Biol. 130, 1189-1196). As the EV22-recognizing sequence LRSG is found in the
integrin Echovirus 22 (EV22)1 was
originally classified as an enterovirus in the family
Picornaviridae, but recent molecular data suggest that it is
a representative of an independent picornavirus genus (1).
Picornaviruses are small non-enveloped RNA viruses that include several
pathogens of man and animals, and their medical and economic importance
has stimulated considerable research activity. These viruses have a
single-stranded mRNA genome, between about 7 and 8.5 kilobases in
length, surrounded by an icosahedral capsid consisting of 60 copies of
four structural proteins VP1-VP4 (for a review, see Ref. 2); however,
in EV22 the maturation cleavage between VP2 and VP4 does not seem to
occur (3). EV22 is known to cause both diarrhea and respiratory
infections, and occasionally the infection is complicated by the
involvement of the central nervous system (4). EV22 infection is common
in childhood and more than 85% of young adults have EV22
antibodies.2
Interestingly, the sequence of EV22 revealed that it carries a
tripeptide motif arginine-glycine-aspartic acid (RGD) in its capsid
protein VP1 (1). Such an RGD motif is involved in multiple biological
recognition reactions; in particular, the cellular matrix proteins
vitronectin and fibronectin use their RGD sequence in binding to
cell-surface receptors known as integrins (for reviews, see Refs. 5 and
6; Table I). Integrins that recognize the RGD motif include Table I.
Alignment of regions of selected viral and cellular polypeptides
containing an RGD sequence
National Public Health Institute,
Division of
Biochemistry,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 chain and the entire LRSGRG hexapeptide occurs in the matrix metalloproteinase 9 (MMP-9), we carried out blocking experiments with anti-integrin and anti-MMP-9 antibodies. EV22
infection could be blocked in cell cultures with anti-
v, -
1, and, to a lesser extent, with anti-MMP-9 antibodies.
These results imply that EV22 recognizes preferentially
v
1-integrin as a cellular receptor and
MMP-9 may also play a role in the cell-surface interactions of the
virus.
5
1,
IIb
3,
v
1,
v
3, and
v
5
subunit combinations. A number of microbes are also able to utilize
this motif in binding to their host cell (Table I). For instance,
during the initiation of adenovirus 2 (7), coxsackievirus A9 (CAV9;
Refs. 8-10), echovirus 22 (EV22; Ref. 3), and foot-and-mouth disease
virus (11) infections, cellular interactions are known to be mediated by the RGD motifs found in viral proteins. It has also been reported that the bacteria species Yersinia (12) and
Bordetella (13) can recognize integrins on the cell
surface.
EV22
RCPNFFFPLPAPKVTSSRALRGDMANLTNQ
EV23
RCPNFFFPLPAPK.PATRKYRGDLATWSDQ
CAV9
PITDTRKDINTVTTVAQSRRRGDMSTLNTH
FMDV
VLATVYNGECRYSRNAVPNLRGDLQVLAQK
Adenovirus
2
NSNAAAAAMQPVEDMNDHAIRGDTFATRAE
Fibronectin
TISGLKPGVDYTITVYAVTGRGDSPASSKP
Vitronectin
CSYYQSCCTDYYAECKPQVTRGDVFTMPED
Fibrinogen
PSRGKSSSYSKQFTSSTSYNRGDSTFESKS
Osteopontin
DLPATEVFTPVVPTVDTYDGRGDSVVYGLR
Bone
sialoprotein
GEYEYTGVNEYDNGYEIYESRGDNYRAYED
During the past few years, several other picornavirus receptors, in
addition to those recognized by CAV9, foot-and-mouth disease virus, and
EV22, have been identified. The cellular receptor of the major group of
rhinoviruses is the intercellular adhesion molecule-1 (14, 15).
Intercellular adhesion molecule-1 and the poliovirus receptor (16) are
members of the immunoglobulin superfamily. Furthermore, it has been
reported that the integrin 2
1 (VLA-2)
acts as a cellular receptor for EV1 (17, 18), whereas several other EVs
interact with the decay accelerating factor (CD55; Refs. 19, 20). At
least six EV serotypes (subtypes 6, 7, 11, 12, 20, and 21) and three
coxsackie-B viruses (subtypes 1, 3, and 5; Refs. 21 and 22) can use
decay accelerating factor as their receptor. A polypeptide belonging to
the nucleolin family is also involved in coxsackie-B viruses
cell-surface interactions (23).
It has been reported previously that CAV9 can compete with the binding of EV22 to green monkey kidney cells (9). This suggests that EV22 and CAV9 may share a common cellular receptor. The aim of the present study was to illustrate further the interactions of these RGD-containing human picornaviruses with the host-cell membrane proteins and to identify receptors interacting with the viruses. For this purpose, the technique of affinity selection of virus-binding peptides from a phage display random peptide library was applied. We have isolated peptide motifs that suggest the involvement of integrins and also, quite unexpectedly, of a proteolytic enzyme MMP-9 in the cellular entry of EV22.
A549, a human lung carcinoma cell line (obtained from the American Type Culture Collection, ATCC), was used for EV22 (1) propagation and plaque tests, and LLC-Mk2 cells (ATCC) were used to grow CAV9 (strain Griggs; ATCC). The viruses were purified in sucrose gradients as described previously (24).
Phage Display Peptide LibrariesWe used a combination of phage libraries containing 5-9 amino acid long cyclic peptides inserted in the pIII capsid protein in the M13 phage. The libraries were constructed using the fuse5 vector (25) as described earlier (26-28).
Selection of EV22-binding Peptides from the Phage Display LibraryIn the first round of biopanning, the purified EV22 or CAV9 was used at a concentration of 5 µg per well in phosphate-buffered saline containing 0.5 mM MgCl2 and incubated overnight at 4 °C in microtiter wells. The wells were then saturated with the blocking solution (0.1 M NaHCO3, 5 mg/ml bovine serum albumin (BSA)) for 1 h at 4 °C. Prior to panning with the virus, the phages were incubated for 1 h at 4 °C in BSA-coated wells in 500 µl of Tris-buffered saline supplemented with 1 mg/ml BSA and 0.5 mM MgCl2. The phage solution was then transferred to the virus-coated wells, and after incubation for 1 h at 4 °C, the unbound phages were removed by washing 10 times with the Tris-buffered saline containing 0.5% Tween 20. The bound phages were eluted with 400 µl of 0.1 M HCl (pH 2.2, adjusted with glycine) in the presence of 1 mg/ml BSA. After the elution, pH was neutralized with 75 µl of 1 M Tris-HCl. The eluted phages were amplified using K91kan bacteria and purified by precipitation with polyethylene glycol as described earlier (25). After amplification, the panning procedure was repeated twice as described above with the exception that the wells were coated with 1 µg of the purified virus. Phage DNA was purified (25) and sequenced using the Sequenase 2.0 kit (U. S. Biochemical Corp.). Peptide sequences found in the phages were compared against protein sequences in the SwissProt data base using the FASTA program (29).
Peptides and AntibodiesThe synthesized peptides were
purified by high performance liquid chromatography. CLRSGRGC was
reduced and alkylated essentially as described (30) and subsequently
purified by high performance liquid chromatography. The following
antibodies were used in the virus blocking experiments: the monoclonal
antibodies included anti-v L230 (ATCC),
anti-
5 P1D6 (Life Technologies, Inc.), and anti-
3 90BB10 B7 (kindly provided by Prof. Ismo
Virtanen, Department of Anatomy, University of Helsinki). Anti-MMP-9
(Ref. 31; kindly provided by Dr. Timo Sorsa, Department of
Periodontology, University of Helsinki) and anti-
1 R322
(a gift from Dr. Jyrki Heino, MediCity Research Laboratory, University
of Turku) were polyclonal rabbit antisera.
The A549 cells were grown as a monolayer in 3.5-cm diameter wells (8). The cells were washed once with Hanks' balanced salt solution supplemented with 20 mM Hepes, pH 7.4 (h-Hanks), and 50 µl of antibodies or peptides (dilutions made in h-Hanks, containing 0.6% fetal calf serum) was added on the cells and incubated for 45 min at room temperature. Fifty µl (approximately 100 plaque-forming units) of EV22 or CAV9 dilution (made in h-Hanks, 0.6% fetal calf serum, containing the corresponding antibody or peptide) was added onto the cells and incubated for 15 min at room temperature. The virus solution in the plates was replaced with 0.5% carboxymethyl cellulose in the culture medium, and the incubation was continued for 48 h in a CO2-humidified incubator at 37 °C. Prior to counting the number of the virus plaques, the cells were stained for 5 min with crystal violet solution (0.25% crystal violet, 2% formaline, 10% ethanol, 0.5% CaCl2, 35 mM Tris).
EV22- and CAV9-binding peptides were selected from phage display peptide libraries containing random peptides from five to nine amino acids, each peptide flanked by cysteine residues. Microtiter wells were coated with the purified viruses, and the phages exhibiting binding activity to the virus were selected by a biopanning protocol.
After three cycles of panning in EV22-coated wells, 170 times more phages could be eluted from the virus-coated wells than from those coated with BSA (control wells). Seventeen phage plaques were selected for further sequence analysis. Twelve of them carried the cyclic peptide CLRSGRGC, and five phages had the CLRSGFGC sequence (Table II). After three rounds of panning in CAV9-coated wells, approximately five times more phage were eluted from the wells containing the virus than from the control wells. Fifteen of the CAV9-binding phages were sequenced. Eleven had a sequence CVWDWGDC, two contained CVWDLGRC, and two CVWDQGIC sequence (Table II). Although the phage libraries used in the study contained peptides of different length, all the sequenced phage carried a hexapeptide. This suggests that the cyclic hexapeptide motifs bind more tightly to the viruses than other peptides in the libraries.
|
The consensus sequences LRSG (EV22) and VWD (CAV9) were compared
against the sequences in the SwissProt data bank
(Table III). Since it is known that
integrins 5
1 and
v
1 recognize the RGD sequence in
fibronectin (32), it is notable that, for instance, the
1 integrin subunit also shares the sequence LRSG in its
extracellular domain (33). A complete identity with the LRSGRG peptide
was found in the matrix metalloproteinase 9 (MMP-9; Ref. 34). It has
been recently shown that MMP-2 is colocalized with integrin
v
3 on the cell surface of invasive cells
(35).
|
Among the VWD-containing proteins is integrin 5 subunit
which contains a VWDQ sequence in its extracellular domain; integrin
v
5 recognizes the RGD sequence in
vitronectin (32).
To study further the
functional role of selected virus-binding peptides in the cell-surface
interactions of EV22 and CAV9, the capability of synthetic peptides to
block the infection was assayed. An RGD-containing peptide representing
the C terminus of EV22 VP1 polypeptide (SRALRGDMANLTNQ) and
an RGD-binding peptide (CWDDGWLC; Ref. 36) were also used in the
blocking experiments. The results of these experiments, obtained by the
plaque tests, are shown in Fig. 1. The
C-terminal EV22 VP1 peptide and the cyclic CLRSGRGC peptide blocked
EV22 infection at a concentration of 0.1 mM, whereas the
control peptide (NGKKKNWKKIM, the N terminus of EV22 VP3) did not
interfere with the initiation of infection at this concentration. The
activity of CLRSGRGC was lost after reduction and alkylation of the
cysteines (not shown). At a concentration of 1 mM, the
RGD-recognizing CWDDGWLC peptide blocked the infection, whereas at
lower concentrations the effect disappeared.
The peptide found in the CAV9-binding phages (CVWDWGDC) had no effect on the EV22 growth. For comparison, blocking experiments with the peptides were also studied in CAV9 infection. Only the RGD-containing peptide blocked the infectivity of this virus. The blocking effect at a peptide concentration of 0.1 mM was even 40% higher, when compared with the EV22 blocking activity. None of the other peptides significantly inhibited CAV9 infection.
Anti-integrin and Anti-MMP-9 Antibodies Block EV22 InfectionSince the consensus sequence LRSG (found in the
EV22-binding phage) is present in the integrin 1 subunit
and in MMP-9, we carried out blocking experiments using anti-integrin
and anti-MMP-9 antibodies (Fig.
2A). The monoclonal
anti-
v antibody and polyclonal serum recognizing the
1 integrin subunit exhibited blocking of the EV22
infection at high dilutions (1:1000), whereas the monoclonal anti-
3 antibody had some effect on the virus growth only
at a dilution of 1:10. The polyclonal anti-MMP-9 antibody inhibited EV22 infection but to a lesser extent than the anti-
v
and -
1 antibodies. The monoclonal anti-
5
antibody used had no effect on the virus growth at any dilution.
The inhibitory effect of the antibodies was also studied in CAV9
infection (Fig. 2B). CAV9 infection was blocked with the anti-v and -
1 antibodies (30 and 60%
inhibition, respectively) at dilutions of 1:100. The
anti-
3 antibody also blocked the virus infection at the
same dilution, although the effect was significantly lower. Some
blocking of CAV9 infection was also seen with the anti-MMP-9 antibody,
but the effect vanished at the antibody dilution of 1:100. As in the
case of EV22, the anti-
5 antibody had no effect on the
initiation of the growth cycle of CAV9.
In this study we have applied an approach based on phage display
peptide libraries to elucidate cell-surface interactions of EV22. We
demonstrate that EV22 infection can be blocked with (i) the CLRSGRGC
peptide found in the EV22-binding phages, (ii) an RGD-containing
peptide representing the C terminus of EV22 capsid protein VP1, and
(iii) an RGD-binding peptide (36). The LRSG consensus sequence, found
in the EV22 binding phages, is present in the extracellular domain of
integrin 1 subunit. Integrins
v
1 and
5
1
are known to bind to the RGD sequence (32). The amino acid sequence
found in the EV22-binding phages is also identical to the sequence in
the matrix metalloproteinase 9 (MMP-9).
To illuminate further the role of integrins and MMP-9 in the
cell-surface interactions of EV22, we carried out blocking experiments with anti-integrin and anti-MMP-9 antibodies. The monoclonal
v antibody and polyclonal
1 antiserum
clearly inhibited the infection, whereas the anti-
3
antibody had a blocking effect only at high concentrations.
Furthermore, anti-MMP-9 antibody also inhibited EV22 infection,
although less efficiently. These results suggest that integrin
v
1 acts preferentially as a cellular
receptor for EV22 and MMP-9 may also be involved in the receptor
interactions of the virus.
CAV9-binding peptides were also selected from the phage display
library, and blocking experiments were performed. Only the RGD-containing peptide clearly blocked the virus infection. Because the
sequence (VWDQ) found in the CAV9 binding phage is present in the
integrin 5 chain, we carried out virus blocking tests with a monoclonal anti-
v
5 antibody, but
it had no effect on the virus growth (data not shown). However, the
CAV9 infection was blocked by the anti-
v monoclonal
antibody and rabbit antiserum recognizing the
1 subunit.
It is possible that the peptide identified by the phage display library
and the RGD-recognizing peptide both bind the RGD-containing motif in
CAV9, but this interaction does not necessarily block infectivity. This
phenomenon has also been observed in experiments where the motif has
been deleted by trypsin treatment (8) or by mutation (10). The
anti-
3 integrin subunit as well as the anti-MMP-9
antibody also had a blocking effect but only when used at the higher
concentrations. Previously published data support the idea that at
least
v
3 integrin plays a role in the
cell-surface interactions of CAV9 (9).
The blocking experiments suggest that EV22 may bind to the
v
1 integrin with the RGD-containing C
terminus of capsid protein VP1. In addition, our results indicate that
at least one of the virus-binding sites is evidently located in the
region of amino acids 103-106 (LRSG) in the
1 chain.
This site is located near the I domain, a region in the
1 subunit that is known to participate in the binding to
the ligand (37). However, the results do not reveal how the
v chain is involved in the virus-receptor interactions. One possibility is that in the experimental conditions the
anti-
v antibody binds to the
v subunit in
such a manner that sterically blocks the binding of EV22 to the
1 chain.
Our results raise the possibility that matrix metalloproteinase 9 (MMP-9) is involved in the cell-surface interactions of EV22 because
the infection could be inhibited by the CLRSGRGC peptide and by
anti-MMP-9 antibody. The LRSGRG sequence is present in the C-terminal
hemopexin domain of MMP-9 which has been shown to be important for the
ability of the protein to dimerize (38). We have also independently
isolated the LRSGXG motif (where X is
preferentially arginine) during panning on purified MMP-9 using the
same phage libraries described
here.3 These results suggest
that the LRSGRG sequence could be involved in the dimerization of
MMP-9, and EV22 may be able to bind MMP-9 through the dimer interface.
It has been shown recently that matrix metalloproteinase 2 interacts
with integrin v
3 on the cell surface (35), and the integrin can simultaneously bind to proteolyzed collagen
fragments. The
v
1 integrin could also
interact with MMP-9, and the complex might then be involved in the
internalization of EV22. Alternatively, EV22 could separately interact
with either
v
1 or MMP-9.
Three human picornaviruses (CAV9, EV22, and EV23) have a functional RGD
motif; in addition, foot-and-mouth disease virus interacts with the
v
3 integrin using the viral RGD sequence
(11, 39). In the three human viruses, the motif is located at the C
terminus of the capsid polypeptide VP1. In CAV9, this region can be
deleted by trypsin treatment (8) or the RGD motif mutated (10) without complete loss of infectivity indicating that the virus can use alternative pathways in its entry into the host cell. The processing of
the C-terminal extension of CAV9 VP1 by proteolytic enzymes may have
implications in the pathogenicity because during the infection in the
gut the virus is prone to the action of intestinal proteases. Whether
destruction of the RGD motif in EV22 abolishes the infectivity is,
however, currently unknown.
EV22 and CAV9 compete for cell-surface binding, and the latter is known
to interact with the vitronectin receptor
(v
3-integrin; Ref. 9). This observation,
suggesting that EV22 would also utilize an RGD-recognizing integrin on
the cell surface, is strongly supported by our findings that peptides
interacting specifically with the virus share sequence homology with
the integrin
1-subunit, and the infection is blocked by
1-antibodies. Although CAV9 and EV22 exhibit
similarities in the cell-surface recognition mechanisms, there are also
clear differences. A monoclonal antibody that is able to block the
infection caused by typical echoviruses (excluding serotypes 22 and 23)
and CAV9 is unable to inhibit EV22 infectivity (40). This suggests that
additional receptor activities can also be important and may be crucial
in the determination of tissue tropism and pathogenesis of these
viruses.
Due to the possibility of rapidly selecting large numbers of peptides that recognize target molecules, the phage display peptide libraries represent a powerful approach in detailed studies of virus-cell interactions. The technique has been successfully used, for example, for the identification of (i) complementary peptide sequences in counterpart proteins of integrins (27, 28, 30), (ii) ligands reacting with the adenovirus 2 penton capsomer (41) and Puumala virus (42), and (iii) epitope(s) recognized by monoclonal antibodies (43, 44) or polyclonal serum (45). In our study, we used the phage peptide library to investigate the receptor interactions of EV22 and CAV9. Although the EV22 peptides obtained by using the phage display technique blocked the infectivity of the virus, the peptides identified in a similar manner by using CAV9 as a target did not inhibit the growth cycle of CAV9. This can be explained either by inefficient binding of the latter peptides or by the differences in cell-surface recognition mechanisms of these two viruses; the RGD-mediated attachment may be the major mechanism in EV22, whereas CAV9 is able to use alternative pathways in its entry as already shown by mutation analysis of the RGD-containing region in CAV9 (10). Our results clarify previously reported receptor specificities of EV22 and propose new, perhaps more complex, mechanisms for EV22 entry.
We thank Drs. Jyrki Heino, Merja Roivainen, Timo Sorsa, and Prof. Ismo Virtanen for providing the antibodies. Drs. Jyrki Heino, Merja Roivainen, Timo Sorsa, Glyn Stanway, and Prof. Tapani Hovi are also acknowledged for stimulating discussions and Marita Maaronen for skillful technical assistance.