INTRODUCTION

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Human parechovirus 1 (HPEV1), a representative of an independent picornavirus genus (19, 24) previously classified as echovirus 22, is a small, nonenveloped, single-stranded RNA virus. Infection of humans, especially infants and young children, can induce respiratory symptoms, encephalitis, and flaccid paralysis (14, 16). HPEV1 carries a tripeptide arginine-glycine-aspartic acid (RGD) motif in its VP1 capsid protein (19, 35), a sequence recognized by v integrins (18, 29). It has been found in previous studies using peptide libraries that HPEV1 possibly utilizes v integrins and preferably v1 as receptors in its infectious cycle (27).

Integrins are a large family of heterodimeric receptors, which appear to be major receptors by which cells attach to extracellular matrices; they also mediate important cell-cell adhesion events (18, 29). Integrins are also involved in a number of tissue remodeling events, such as wound repair and bone resorption (12, 15). Integrin-ligand interactions mediate the activation and regulation of intracellular signaling pathways within cells, which control transcriptional and ligand binding functions (31, 34). The RGD sequence which is present in many integrin natural ligands (vitronectin, fibronectin, fibrinogen, etc.) is recognized by specific cellular integrins such as v3, v5, v1, IIb3, and 51 (18, 29, 30).

Integrins have been also subverted by a number of bacterial pathogens such as Lyme disease spirochetes (9) and Bordetella pertussis (20), viral pathogens such as rotaviruses (10) and papillomaviruses (13), and also members of the Picornaviridae family. The latter include echoviruses 1, 8, and 9, which utilize integrins as receptors (4, 5, 38). Coxsackievirus A9 and foot and mouth disease virus, which both exhibit an RGD sequence found in the VP1 capsid protein (1, 7, 8), use integrin v3 as a receptor molecule (6, 22, 23, 25, 26, 28, 37).

In this study, we investigated the requirements for HPEV1 attachment to cells and have shown that both integrin v3 and integrin v1 are directly involved in HPEV1 attachment by acting as the virus binding receptors in the viral infectious cycle.

	MATERIALS AND METHODS

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Cell lines. The human lung carcinoma (A549) cell line was maintained in minimal essential medium containing 1% nonessential amino acids, 10% heat-inactivated fetal bovine serum, and 100 µg of gentamicin per ml.

HPEV1 plaque assay. For the production of virus plaques, the cells were infected with virus, and a plaquing overlay was used. The overlay consisted of the appropriate medium to which 0.5% (wt/vol) carboxymethyl cellulose was added. The HPEV1 plaque assays were also repeated without the presence of overlay. Plaques were visualized by staining with 0.2% (wt/vol) crystal violet in 1% (vol/vol) ethanol.

Antibodies and ligands. Monoclonal antibodies (MAbs) LM609 (a function-blocking MAb specific for integrin v3), 6S6 (a function-blocking MAb specific for integrin 1), B3B11 (specific for integrin 1), and P1F6 (a function-blocking MAb specific for integrin v5) were obtained from Chemicon, as were VNR139 (v-chain-specific MAb) and BHA2.1 (MAb specific for integrin 21). MAbs NK1-M9 (v specific) and the Y2/51 (3-chain specific) were obtained from Zymed Laboratories. Rabbit polyclonal sera specific for integrins 2 (AB1944), 5 (AB1928), 4 (AB1922), and 5 (AB1926) were obtained from Chemicon. HPEV1 neutralizing monkey polyclonal serum was obtained from the American Type Culture Collection. Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig) and HRP-conjugated goat anti-rabbit Ig were obtained from Kirkegaard & Perry Laboratories and Antibodies Incorporated, respectively. Normal monkey serum was obtained from Antibodies Incorporated. Vitronectin and fibronectin were obtained from Sigma.

Virus infectivity assays in the presence of integrin natural ligands. Cell lines A549, CHO-v3, CHO-v1, and CHO-wt were grown as a monolayers in six-well plates (Nunc) and incubated with integrin natural ligands (10 to 80 µg/ml) in serum-free medium at room temperature for 50 min. Approximately 250 PFU of HPEV1 particles was added to each culture and incubated at room temperature for 50 min. The monolayer was washed with culture medium and overlaid with 0.5% (wt/vol) carboxymethyl cellulose in culture medium. The incubation was continued for 48 to 72 h in a 7% CO₂ humidified incubator before plaque visualization with crystal violet. Control plates with isotype control IgG were similarly treated.

Virus blocking assays. A549, CHO-v3, CHO-v1, and CHO-wt cells were grown as a monolayer in six-well plates (Nunc). MAbs (2.5, 5, 10, and 15 µg) were added in 1 ml of serum-free medium, the mixture was incubated at room temperature for 50 min, approximately 250 PFU of HPEV1 virus particles was added, and the mixture was incubated at room temperature for 50 min. The monolayer was washed with culture medium and overlaid with 0.5% (wt/vol) carboxymethyl cellulose in culture medium. Incubation was continued for 48 to 72 h in a 7% CO₂ humidified incubator before plaque visualization with crystal violet. Control plates with isotype control IgG were similarly treated.

Labeling of cell surface with NHS-biotin. A549, CHO-v3, CHO-v1, and CHO-wt cells were surface labeled with biotin, using 40 µl of 0.1 M membrane-impenetrable NHS (N-hydroxysuccinimide ester derivative)-biotin reagent (Amersham) in 2 ml of phosphate-buffered saline (PBS) per 10⁸ cells. After 30 min, the reaction was stopped with 1 mM ethanolamine in PBS. Cells were washed three times with PBS and lysed in lysis buffer (1% digitonin, 15 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, 2 mM phenylmethylsulfonyl fluoride).

Immunoprecipitation protocols. A549, CHO-v3, CHO-v1, and CHO-wt cells were surface labeled with NHS-biotin and lysed in lysis buffer as described above. The lysate was precleared with normal monkey serum followed by the addition of 10% (wt/vol) protein A-Sepharose beads (Pharmacia Biotech, Uppsala Sweden) to remove nonspecific binding material. Virus receptor complexes were immunoprecipitated by the addition of 1.5 × 10⁶ PFU of virus; after incubation for 1 h at room temperature, 2 µg of HPEV1-specific monkey serum was added for 1 h at 4°C. The resulting immune complexes were isolated with 10% protein A-Sepharose beads.

Western blotting. Immunoprecipitates were separated by SDS-PAGE and transferred onto a nitrocellulose filter (Schleicher & Schuell, Dassel, Germany) or Immobilon P membranes (Millipore). After transfer, the membrane was immersed for 1 h in blocking solution (5% low-fat dried milk dissolved in 0.1% PBS-Tween) and washed with 0.1% PBS-Tween (two rinses, a 15-min wash, and two 10-min washes). The membrane was then incubated with streptavidin-HRP conjugate or an appropriate dilution of MAbs, followed by 1 h of incubation with a dilution of HRP-conjugated goat anti-mouse Ig or HRP-conjugated goat anti-rabbit Ig. The optimum antibody concentration was determined by dot blot assay (data not shown). After extensive washing with 0.1% PBS-Tween, the antigen was visualized by the enhanced chemiluminescence procedure (Amersham) according to the manufacturer's instructions.

	RESULTS

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HPEV1 displays an RGD motif in the VP1 capsid protein (19, 35), suggesting integrins as candidate receptors for this virus. To analyze the involvement of integrins in HPEV1 attachment, we used A549 cells, which are susceptible to HPEV1 infection. To determine the presence of v integrins on these cells and to obtain relative semiquantitive information about these integrins, flow cytometric analysis using fluorescein isothiocyanate (FITC)-conjugated antibodies was used. An integrin v3-specific MAb (LM609), a 1-specific MAb (6S6), and an integrin v5-specific MAb (P1F6), which were titrated on these cells to determine the optimum concentration of each antibody (data not shown), were used. Our results showed that these cells express integrins v3, v1, and v5 (Fig. 1). However, integrins v3 and v1 (Fig. 1B and C) were more abundant than integrin v5 (Fig. 1D).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Flow cytometric analysis of integrin v3, v1, and v5 expression in A549 cells. Control A549 cells were incubated with FITC-conjugated rabbit anti-mouse IgG (A), integrin v3-specific MAb LM609 (B), integrin 1-specific MAb 6S6 (C), and integrin v5-specific MAb P1F6 (D). The histograms display relative cell numbers as a function of relative fluorescence intensities.

To investigate whether integrins are HPEV1 receptors, we performed blocking experiments using MAbs specific for v integrins that are known to recognize the RGD motif in ligands and counterreceptors and a MAb specific for integrin 21 which recognizes the aspartic acid-glycine-glutamic acid-alanine sequence instead of the RGD motif (18, 29, 30). Therefore, an v3-specific function-blocking MAb (LM609), an v-specific MAb (NK1-M9), a 1-specific MAb (6S6), an v5-specific MAb (P1F6), and an 21-specific MAb (BHA2.1) were used (Fig. 2 and 3). The results showed that at concentrations 10 µg and above, the v-specific MAb (NK1-M9) inhibited infection by 80%, whereas the v3-specific MAb (LM609) inhibited infection by 65% (Fig. 2). The 1-specific MAb (6S6) inhibited infection by 50%, while the integrin v5-specific MAb (P1F6) inhibited infection by 10% (Fig. 2). The isotype control MAb had no effect on the virus infection (Fig. 2 and 3). A combination of MAbs LM609 and 6S6, used to saturate integrin v3 and 1 receptors, inhibited virus infection by 85% (Fig. 3), whereas a combination of LM609 and P1F6, to saturate integrin v3 and v5 receptors, inhibited infection by 65%. A combination of LM609, 6S6, and NK1-M9 completely inhibited virus infection (Fig. 3). These results show that the v5- and 21-specific MAbs had no significant effect on HPEV1 infectivity, thus leading us to believe that HPEV1 preferentially utilizes integrins v3 and v1.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Percent inhibition of HPEV1 infectivity to A549 cells in the presence of LM609 (v3-specific MAb), NK1-M9 (v-specific MAb), 6S6 (1-specific MAb), P1F6 (v5-specific MAb), BHA2.1 (21-specific MAb), and isotype control MAb at concentrations of 2.5, 5, 10, and 15 µg. The error bars are calculated from the standard deviation over a number on independent experiments.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Percent inhibition of HPEV1 infectivity to A549 cells in the presence of combinations of MAbs at concentrations of 2.5, 5, 10, and 15 µg. For identities of the MAbs, see the legend to Fig. 2. The error bars are calculated from the standard deviation over a number on independent experiments.

Vitronectin and fibronectin are cell matrix proteins and natural ligands for specific cell surface integrins including integrins v1, v3, and v5 (18, 29, 30). Vitronectin and fibronectin, separately or in combination, were added to A549 cells before the addition of HPEV1 particles (Fig. 4) to determine whether they could block infectivity. Our results showed that infectivity was inhibited 70% by vitronectin (Fig. 4), 40% by fibronectin, and 90% by a combination of vitronectin and fibronectin. Since v integrins are known to bind vitronectin, fibronectin, or both, these studies indicate that occupancy of v integrins by cell matrix proteins significantly reduces the susceptibility to HPEV1 infection.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Percent inhibition of HPEV1 infectivity to A549 cells in the presence of different concentrations (10 to 80 µg/ml) of vitronectin, fibronectin, laminin, and a combination of vitronectin and fibronectin. The error bars are calculated from the standard deviation over a number on independent experiments.

To verify the involvement of integrins v1 and v3 as HPEV1 receptors, CHO-v1 (CHO cells transfected and expressing human integrin v1) and CHO-v3 (CHO cells transfected and expressing integrin v3) and CHO-wt cells were used. The results showed that CHO-v1 cells express integrin v1 (Fig. 5E) but not integrin v3 (Fig. 5F). CHO-v3 cells expressed integrin v3 (Fig. 5B) but not v1 (Fig. 5C), whereas CHO-wt cells expressed neither integrin v1 (Fig. 5H) nor integrin v3 (Fig. 5I).

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Flow cytometric analysis of integrin v3 and v1 expression on CHO-v3, CHO-v1, and CHO-wt cells. Control CHO-v3 (A), CHO-v1 (D), and CHO-wt (G) cells were incubated with FITC-conjugated rabbit anti-mouse IgG. To test integrin expression on CHO-v3 cells, integrin v3-specific MAb LM609 (B) or integrin 1-specific MAb 6S6 (C), followed by FITC-conjugated rabbit anti-mouse IgG, was added to the cells. To CHO-v1 cells, integrin 1-specific MAb 6S6 (E) or integrin v3-specific MAb LM609 (F), followed by FITC-conjugated rabbit anti-mouse IgG, was added. To test integrin expression on CHO-wt cells, specific MAb LM609 (I) or specific 6S6 (H) was added, followed by FITC-conjugated rabbit anti-mouse IgG. The histograms display relative cell numbers as a function of relative fluorescence intensities.

CHO-wt cells were tested and found not to be infected by HPEV1 (Fig. 6C). Our experiments with the CHO transfectants showed that the virus could successfully infect CHO-v1 (Fig. 6A) and CHO-v3 (Fig. 6B) cells. To exclude the possibility that CHO-wt cells were infected by the virus but no plaques were formed, HPEV1 particles (10⁷ PFU) were added to CHO-wt (10⁶) cells and also A549 (10⁶) cells as a control. These cells were incubated at different time periods; for each time period, the cells were frozen and thawed to release HPEV1 particles that may have been produced. The cell lysate was added to A549 cells, which were then assayed for the presence of virus by plaque formation. The data showed no plaque formation on A549 cells when CHO-wt lysate had been added. In contrast, plaques formed on A549 cells when A549 lysate had been added (data not shown). The A549 cells were killed within 10 h, while the CHO-wt cells were incubated for up to 96 h without the formation of plaques.

View larger version (132K): [in this window] [in a new window]   FIG. 6.   Results of HPEV-1 plaque assay on CHO-v1 (A), CHO-v3 (B), and CHO-wt (C) cells. The plates are representative of a number of independent experiments.

The results of blocking experiments performed with v (NK1-M9)- and 1 (6S6)-specific MAbs showed that this combination of antibodies completely inhibited virus infection of CHO-v1 cells. Also, the integrin v3 MAb (LM609) completely inhibited virus infection of CHO-v3 cells (Fig. 7). We found no effect on infectivity of CHO-v1 and CHO-v3 cells when isotype control MAbs were used (Fig. 7).

View larger version (30K): [in this window] [in a new window]   FIG. 7.   Percent inhibition of HPEV1 binding to CHO-v1 and CHO-v3 cells in the presence of a combination of v-specific MAb NK1-M9 and 1-specific MAb 6S6 (black bars), in the presence of an isotype control MAb (clear bars), in the presence of v3-specific MAb LM609 (striped bars), and in the presence of an isotype control MAb (black and white bars). The error bars are calculated from the standard deviation over a number of independent experiments.

To test whether HPEV1 utilizes any cell surface molecules in its infectious cycle other than the integrins mentioned above, A549 cells, which are susceptible to HPEV1 infection, were used for immunoprecipitation experiments. A549 cell lysate was incubated with virus particles followed by the addition of HPEV1-specific neutralizing serum and protein A-Sepharose beads. SDS-PAGE analysis of the immunoprecipitated material revealed the presence of 120- and 100-kDa bands (Fig. 8G); a faint band of 20 kDa was also visible after extended exposure (data not shown). No proteins were detected in the absence of virus particles (Fig. 8E) or when an irrelevant antiserum was used (Fig. 8F). Western blotting was used to determine the identity of these bands; a panel of integrin - and -chain-specific antibodies, MAbs VNR139 (v specific) and Y2/51 (3 chain specific), revealed that these bands corresponded to the v and 3 chains of integrin v3 (Fig. 8M and N). When CHO-v1, CHO-v3, and CHO-wt biotinylated cell surface lysates were used for immunoprecipitation experiments with HPEV1 particles, Western blotting using a panel of integrin-chain-specific antibodies (data not shown) demonstrated that integrin v1 was immunoprecipitated by the virus particles from CHO-v1 cells (Fig. 8A), whereas when CHO-v3 cell lysate was used, integrin v3 was immunoprecipitated by HPEV1 particles (Fig. 8D). When CHO-wt cell lysate was used, no proteins were immunoprecipitated by HPEV1 particles (Fig. 8B).

View larger version (68K): [in this window] [in a new window]   FIG. 8.   SDS-PAGE of immunoprecipitated HPEV1 receptor complexes under reducing conditions. Cell surface-biotinylated A549 cells were solubilized in 1% digitonin and immunoprecipitated with HPEV1 virions followed by HPEV1-specific monkey neutralizing serum (G), or in the absence of HPEV1 virions, with HPEV1-specific monkey neutralizing serum alone (E), with an irrelevant antiserum (F), or with normal monkey serum (C). As controls, cell surface-biotinylated CHO-v1 (A), CHO-v3 (D), and CHO-wt (B) were solubilized in 1% digitonin and immunoprecipitated with HPEV1 virions followed by HPEV1-specific monkey neutralizing serum. The membrane from the A549 cell lysate immunoprecipitations was Western blotted with v-chain-specific MAb VNR139 (M), with 3-chain-specific MAb Y2/51 (N), with 1-chain-specific MAb B3B11 (H), and with rabbit polyclonal sera specific for integrins 2 (I), 4 (J), 5 (K), and 5 (L), followed by either HRP-conjugated goat anti-mouse Ig or HRP-conjugated goat anti-rabbit Ig. The positions of molecular weight markers are shown to the right.

	DISCUSSION

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Worldwide HPEV1 infections are very common, causing mainly respiratory and gastrointestinal symptoms (16); in rare cases, HPEV1 is also responsible for the more serious and life-threatening disease encephalitis as well as flaccid paralysis (14, 16). Receptor-virus associations are the initial step of a viral infection. Previous studies using phage display peptide libraries to identify the HPEV1 receptor molecules have shown that the virus binds peptides containing amino acid motifs found in the v1 integrin (27). In this study, we attempted to identify molecules involved in HPEV1 binding. To this end, we performed blocking experiments with a panel of MAbs specific for integrins v3, v1, and v5; as a control, we used an integrin 21-specific MAb, since it does not recognize the RGD sequence displayed on natural ligands. Our results showed that the 21-specific MAb had no effect whereas the v5-specific MAb had a very minor effect on virus infection. The v3 MAb showed a 65% inhibition. A combination of v5 and v3 MAbs reduced infectivity by 65%, the same effect as for the v3 MAb; a combination of 1 and v3 MAbs could inhibit virus infection by 85%, and a mixture of 1, v, and v3 MAbs completely inhibited virus infection. Thus, these data suggest that whereas integrins v1 and v3 play an important role in HPEV1 infection, integrin v5 is not involved in this virus infectious cycle.

To verify the use of v integrins by HPEV1, integrin v natural ligands, such as fibronectin and vitronectin, were used. The results showed that vitronectin and fibronectin reduced virus infection, while a combination of the two ligands inhibited infection by 90%, thus verifying that HPEV1 utilizes v integrins.

To confirm our finding that the virus could utilize v integrins, specifically integrins v3 and v1, we tested whether HPEV1 could bind on cell surface integrin v1 and also v3. To achieve this, CHO-v1 and CHO-v3 cells expressing integrins v1 and v3, respectively, were used. The experiments showed that the cell lines could be successfully infected by HPEV1, thus confirming that the virus can bind on integrins v3 and v1.

Immunoprecipitation experiments using cell surface-labeled A549 cell lysate and HPEV1 particles were performed to see whether the virus utilizes receptor molecules other than integrins v3 and v1. The results showed that the virus could immunoprecipitate a 100/120-kDa heterodimer which was identified as integrin v3 by Western blotting with a panel of integrin-chain-specific antibodies. The 3 chain seems to be more intensely labeled than the v chain, possibly due to the labeling procedure. This chain may express more lysine residues than the  chain; since NHS-biotin (our labeling reagent) labels lysines, the 3 chain might be more heavily labeled. Results of immunoprecipitation experiments performed with cell surface-labeled cell lysates showed that HPEV1 could immunoprecipitate integrin v3 from CHO-v3 cell lysate, integrin v1 from CHO-v1 cell lysate, and no protein from CHO-wt lysate, leading us to believe that HPEV1 utilizes only integrins v1 and v3.

Overall, we found that HPEV1 can utilize efficiently both integrin v3 and integrin v1 as receptor molecules, making its infectious cycle more efficient by virtue of the ability to alternate receptors. In this respect HPEV1 is like the coxsackie B viruses, which can use either decay-accelerating factor (3, 33), a 100-kDa nucleolin-related protein (11), or coxsackievirus-adenovirus receptor protein (2) as receptor molecules, as well as measles virus, which can utilize both CD46 and moesin (32). Another example is encephalomyocarditis virus, which can use either the Ig vascular cell adhesion molecule (17) or a 70-kDa cell surface sialoglycoprotein (21).

Although it has been shown that HPEV1 can bind both integrins, we found that in A549 solubilized cell extract the virus binds integrin v3; this could be explained by the fact that the virus interacts initially with v3 and then with v1, or preferentially in the presence of both integrins utilizes v3. We therefore conclude that HPEV1 binds both integrins as receptor molecules, but in cells which express both v3 and v1, it may have a higher affinity for integrin v3.