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Originally published In Press as doi:10.1074/jbc.M410208200 on October 7, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54533-54541, December 24, 2004
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Interaction of West Nile Virus with {alpha}v{beta}3 Integrin Mediates Virus Entry into Cells*

Justin Jang-hann Chu and Mah-Lee Ng{ddagger}

From the Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597

Received for publication, September 7, 2004 , and in revised form, October 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The functional receptor for the flavivirus West Nile (WNV) infection has been characterized in this study with a combination of biochemical and molecular approaches. A 105-kDa protease-sensitive glycoprotein that binds WNV was isolated from the plasma membrane of cells permissive to WNV infection. The protein was subjected to peptide sequencing, and this glycoprotein was identified as a member of the integrin superfamily. Infection of WNV was shown to be markedly inhibited in Vero cells pretreated with blocking antibodies against {alpha}v{beta}3 integrin and its subunits by receptor competition assay. It was also noted that cells pretreated with antibodies against {alpha}v{beta}3 integrin can effectively inhibit flavivirus Japanese encephalitis but to a lesser extent flavivirus dengue infections. West Nile virus entry is independent of divalent cations and is not highly blocked by arginine-glycine-aspartic acid (RGD) peptides, suggesting that the interaction between the virus and {alpha}v{beta}3 integrin is not highly dependent on the classical RGD binding motif. In addition, gene silencing of the {beta}3 integrin subunit in cells has resulted in cells largely resistant to WNV infection. In contrast, expression of recombinant human {beta}3 integrin substantially increased the permissiveness of CS-1 melanoma cells for WNV infection. Soluble {alpha}v{beta}3 integrin can also effectively block WNV infection in a dose-dependent manner. Furthermore, WNV infection also triggered the outside-in signaling pathway via the activation of integrin-associated focal adhesion kinase. The identification of {alpha}v{beta}3 integrin as a receptor for WNV provides insight into virus-receptor interaction, hence creating opportunities in the development of anti-viral strategies against WNV infection.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
West Nile virus (WNV)1 is a small enveloped virus of the Flaviviridae family. West Nile virus is the causative agent of the disease syndrome named West Nile Fever including a spectrum of associated complications (meningo-encephalitis and acute flaccid paralysis) (1). This is a re-emerging arthropod-borne disease that is responsible for recent large outbreaks in the Western hemisphere. In 2002, there were 4,156 human infections and 284 deaths reported in the United States (2). Currently, there is no vaccine or antiviral agent against this pathogenic virus.

The WNV has a single-stranded, positive-sense RNA molecule of ~11 kb in size (3). The RNA genome encodes for three structural proteins (capsid, precursor of membrane, and envelope) and seven non-structural proteins that are essential for intracellular replication (4). The lipid bilayer envelope of the WNV (which encloses the nucleocapsid) is derived from host cell membrane and is modified by insertion of the precursor membrane and the viral envelope protein (5).

Several studies have documented that the envelope protein of flaviviruses (which is exposed on the surface of the envelope membrane) is the virus attachment protein and is involved in the interaction with the cellular receptor molecule (6, 7). Crystallography data on the ectodomain of the flavivirus envelope protein reveals three distinct domains: a central domain designated as domain I, an elongated dimerization region designated as domain II, and domain III, having an immunoglobulin-like constant domain (8). Both domain II and III of the envelope protein have been suggested to be important for binding to the cellular receptor (9–12).

Previous studies have documented the involvement of different glycoproteins or lipoproteins of unknown identities as the putative receptors on various cell types for entry of flaviviruses (13–15). It has been shown that for dengue virus (DV), heparin sulfate is required for the initial binding of the virus to the cell surface, but additional high affinity receptors are necessary to mediate virus internalization (7). Alternatively, dendritic cell-specific ICAM-3 grabbing non-integrin expressed on the surface of dendritic cells has recently been shown to mediate entry of DV (15, 16) but is not necessary for WNV and yellow fever virus (15). So far, the functional receptor molecule(s) that is responsible for entry of WNV has been elusive. This study embarked on the characterization of the functional receptor responsible for WNV infection. Our data implicate the {alpha}v{beta}3 integrin, a prominent endothelial cell receptor, as the functional receptor and the associated signaling pathway necessary for WNV entry into vertebrate cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Viruses—Vero cells (America Type Culture Collection (ATCC), Manassas, VA) were maintained in Medium 199 containing 10% inactivated fetal calf serum. Chick embryo fibroblast cells were prepared from 9-day chick embryos in the laboratory and were maintained in M199 with 20% fetal calf serum. The CS-1 and CS-1{beta}3 melanoma cells (kind gifts from Dr. D. Cheresh, Scripps Research Institute), HeLa cells, baby hamster kidney, HepG2, N2A, and U937 (ATCC) were grown in RPMI 1640 medium containing 10% fetal calf serum. All viruses were generous gifts from Prof. Edwin Westaway. Flavivirus, West Nile (Sarafend), Japanese encephalitis virus (JEV) (Nakayama), and dengue virus serotype 2 (New Guinea) were propagated essentially as described previously (17). Unlabeled and [35S]methionine-labeled flaviviruses were concentrated and purified by sucrose gradient centrifugation as described previously (17). Homogenous population of infectious WNV particles was obtained after purification. The morphological changes of virus-infected cells were visualized using an Olympus IX81 microscope equipped with differential interference contrast mode.

Receptor Competition and Virus Entry (Binding and Penetration) Assay—Cells were incubated with different concentrations (0.25–25 µg/ml) of functional blocking integrin antibodies ({alpha}1-FB12; {alpha}2-P1E6; {alpha}3-P1B5; {alpha}4-P1H4; {alpha}5-P1D6; {alpha}6-NKI-GoH3; {alpha}v-P3G8; {beta}1-P4G11; {beta}2-P4H9; {beta}3-25E11; {beta}4-ASC-9; {beta}5-{alpha}v{beta}3-LM609; {alpha}v{beta}5-P1F6) (Chemicon International), {alpha}v{beta}3, vascular adhesion protein-1, MPTP{Delta}, spectrin {beta} chain, protocadherin (Santa Cruz Biotechnology), integrin ligands (fibronectin, vitronectin, heparin, chondroitin sulfate, laminin, collagen type 1) (Sigma), BSA (Invitrogen), and RGD peptides (Sigma) in M199 for 30 min at 4 °C followed by the addition of [35S]methionine-labeled flaviviruses at a multiplicity of infection (m.o.i.) of 10. To assay for the virus binding to the cell surface, the virus was incubated with the cells for 1 h at 4 °C (low temperature treatment of cells immobilized endocytotic activities at the plasma membrane), and to assess virus penetration, the virus was incubated with the cells for 1 h at 37 °C. Excess or unbound virus was inactivated with acid citrate buffer (pH 3.0) and removed by extensive washing with phosphate-buffered saline. The cells were then lysed with 1% SDS, and the specific radioactivity was determined. The results are recorded as percentage of inhibition of virus binding or penetration and was calculated based on the radioactivity detected from cells incubated with antibodies/ligands and compared with the untreated cells. Three independent sets of experiments were carried out for each of the antibodies/ligands used. The viability of cells was checked by trypan blue staining to ensure that the concentrations of the antibodies and ligands used in this study are non-cytotoxic.

Inhibition of Virus Entry with Soluble Integrin—West Nile virus (500 plaque-forming units) was first incubated with soluble {alpha}v{beta}3 and {alpha}v{beta}5 integrin (Chemicon International) at 1–10 µg/ml for 1 h at 4 °C. The complex was then added to Vero cell monolayer for1hat37 °C.The cell monolayers were washed with acid citrate buffer (pH 3.0) followed by washing with phosphate-buffered saline three times. The virus-infected cells were overlaid with overlay medium for 4 days at 37 °C. Virus plaques were stained with 0.5% crystal violet/25% formaldehyde solution for 30 min at room temperature.

Virus Overlay Protein Blot Assay—Plasma membrane proteins of Vero cells were prepared and purified as described previously (17). In brief, 80 µg of the plasma membrane proteins were separated by 10% SDS-PAGE and electrophoretically transferred onto nitrocellulose membrane (Bio-Rad). The nitrocellulose membrane was soaked overnight in a milk buffer (5% skimmed milk and 0.5% BSA) before rinsing with phosphate-buffered saline (three times) and incubated with anti-{alpha}v{beta}3 or anti-{alpha}v{beta}5 integrin antibodies (25 µg/ml). This was followed by incubation with the purified radiolabeled WNV. Nonspecific binding of the virus particles was reduced by washing with a high salt buffer wash as described previously (17). The presence of virus binding was detected by exposing the membrane to Kodak film.

Gene Silencing of Integrin {beta}3—Human {beta}3 integrin sequence (5'-AAACATCAATTTGATCTTTGC-3') was cloned into small interfering RNA (siRNA) expression vector pSilencer 3.0-H1 (Ambion) according to the manufacturer's instructions. Appropriate concentrations of pSilencer against {beta}3 integrin and glyceraldehyde-3-phosphate dehydrogenase (internal control) genes and pSilencer (negative control) were transfected into HeLa cells using LipofectaminePlus reagents from Invitrogen according to the manufacturer's instructions. Down-regulation of cell surface {alpha}v{beta}3 integrin was determined by Western blotting and flow cytometric analysis. Cell clones with down-regulated expression of {alpha}v{beta}3 integrin and glyceraldehyde-3-phosphate dehydrogenase were selected by FACS for virus entry assay.

Detection of Integrin Expression by Flow Cytometric Analysis—Suspension cells and single cell suspensions of adherent cells (5 x 106) were washed and incubated with antibodies against integrins at 4 °C for 30 min. The cells were washed and incubated with fluorescein isothiocyanate-conjugated secondary antibodies at 4 °C for 30 min, washed again, and analyzed in a FACScan flow cytometer (BD Biosciences) with the appropriate gating parameter. An isotype control was also included. Dead cells stained with propidium iodide were excluded from the analysis.

Analysis of WNV-induced Focal Adhesion Kinase (FAK) Activation— Vero cells were first serum-starved overnight and subjected to WNV infection at an m.o.i. of 10. At the appropriate timings (5, 10, 20, 30, and 45 min) after the inoculation of WNV onto the serum-starved cells at 37 °C, the cells were washed and lysed with lysis buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.4 mM phenylmethylsulfonyl fluoride, and 0.8% sodium deoxycholate in 0.01 M Tris-HCl, pH 7.4). The cell lysate was spun at 1000 x g for 5 min to remove the nuclei followed by SDS-PAGE and Western blotting as described previously (17). The activation of FAK was detected using antibodies specific for the phosphorylated residue Tyr397 of FAK.

Immunofluorescence Assay—The co-localization of activated FAK and vinculin was detected using immunofluorescence assay. Serum-starved Vero cells were inoculated with WNV at an m.o.i. of 10 for a period of 5 min at 37 °C followed by fixation with ice-cold absolute methanol. The cells were then processed for double immunostaining with antibodies against FAK and vinculin using the method essentially described in Ref. 17. Lysophosphatidic acid (LPA, 200 ng/ml) was used as a positive control to induce the activation of FAK in serum-starved cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Antibodies against {alpha}v{beta}3 Integrin and Subunits Prevent WNV Entry—Recently, we have characterized a 105-kDa protease-sensitive N-linked glycoprotein as the putative receptor for WNV from permissive cell lines (Vero and murine neuroblastoma cells, N2A) (17). In this study, trypsin digestion and mass spectrometry was first carried out to determine the identity of the 105-kDa plasma membrane-associated glycoprotein from Vero cells. The peptide sequencing data suggested a list of putative proteins (Table I). A receptor competition assay was designed according to Ref. 19 for assessing virus entry by incubating Vero cells with polyclonal antibodies against these putative proteins. This served as a preliminary screen to identify the possible protein that mediates WNV entry into Vero cells. Fig. 1 shows that polyclonal antibody against {beta}3 integrin (100 µg/ml) significantly blocked the entry of [35S]methionine-radiolabeled WNV by 75%. In contrast, polyclonal antibodies that were specific for the rest of the proteins had minimal effect on blocking WNV entry into Vero cells. Moreover, the molecular mass of the WNV-binding 105-kDa membrane protein has the closest match to the human {beta}3 integrin subunit.


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  		TABLE I
Peptide sequencing of the 105-kDa membrane protein A number of putative proteins were revealed with matching identities to the 105-kDa membrane protein using the MS-Fit search program.

 



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  		FIG. 1.
The use of antibodies against putative receptor molecules to block West Nile virus entry. The percentage of inhibition of WNV entry is plotted against concentrations of antibodies used. Vero cells treated with polyclonal antibody against {beta}3 integrin shows the highest inhibition of WNV infection, whereas the rest of the antibodies against vascular adhesion protein-1 (VAP), MPTP{Delta} (MPTP), spectrin {beta}1, and protocadherin (PC) have minimal effect on WNV infection.

 
Given this finding, we attempted to further determine the specific integrin molecule or its subunits that mediate binding and entry of WNV. Receptor competition assay was again employed. Vero cells were pre-incubated with a panel of functional blocking antibodies (a range of concentration at 0.25–25 µg/ml) against the subunits of the integrins ({alpha}1, {alpha}2, {alpha}3, {alpha}4, {alpha}5, {alpha}6, {alpha}v, {beta}1, {beta}2, {beta}3, {beta}4, {beta}5) (Chemicon), and WNV binding as well as penetration into Vero cells were determined.

Radioactive [35S]methionine-labeled WNV (m.o.i. of 10) was added to the anti-integrin antibody pretreated cells and further incubated for 1 h at 4 °C (to assay for virus binding to cells) or 1 h at 37 °C (to assay for virus penetration into cells). Excess and unbound virus particles were inactivated, and the cells were washed before radioactivity determination assay. Obvious dose-dependent inhibition of WNV binding and entry was observed for Vero cells pretreated with functional antibodies against {beta}3 and {alpha}v integrin subunits. Functional blocking antibody against {beta}3 integrin exhibited the most significant inhibition of WNV binding (>60%) (Fig. 2a) and penetration (>75%) (Fig. 2b) into cells. As shown in Fig. 2, the antibody against the {alpha}v integrin subunit reduced WNV binding by 50% (Fig. 2c), and penetration was inhibited by >60% (Fig. 2d). Therefore, the data suggested the involvement of both {alpha}v and {beta}3 integrin subunits in both binding and penetration of the virus. Similar isotype-specific antibodies against integrin subunits ({alpha}1, {alpha}2, {alpha}3, {alpha}4, {alpha}5, {alpha}6, {beta}1, {beta}2, {beta}4, {beta}5) did not show any marked inhibition (<10%) of WNV binding and penetration (Fig. 2).



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  		FIG. 2.
Antibodies to {alpha}v{beta}3 integrin and its subunits inhibit WNV binding and penetration. Blocking antibody against {beta}3 integrin subunits inhibits WNV binding (a) to the cell surface as well as virus penetration (b). Anti-{alpha}v integrin antibody inhibits WNV binding (c) and penetration (d) to cell surface. e, virus overlay protein blot assay was performed, and binding of WNV to the 105-kDa membrane protein band is detected (lane 2). Antibody to {alpha}v{beta}3 integrin (lane 1) blocks the binding of WNV to the 105-kDa membrane protein band, whereas the antibody to {alpha}v{beta}5 integrin is not as effective in blocking virus binding (lane 3). f, WNV and JEV entries are significantly inhibited by the functional blocking antibody against {alpha}v{beta}3 integrin but not DV entry. No cytotoxicity is detected for the concentration spectra of antibodies used. Results of three independent experiments (each data point represents the mean ± S.D. of results) are shown. INTA1, {alpha}1 integrin; INTA2, {alpha}2 integrin; INTA3, {alpha}3 integrin; INTA4, {alpha}4 integrin; INTA5, {alpha}5 integrin; INTA6, {alpha}6 integrin; INTAV, {alpha}v integrin; INTB1, {beta}1 integrin; INTB2, {beta}2 integrin; INTB3, {beta}3 integrin; INTB4, {beta}4 integrin; INTB5, {beta}5 integrin; INTAVB3, {alpha}v{beta}3 integrin; INTAVB5, {alpha}v{beta}5 integrin.

 
To further define whether {alpha}v{beta}3 integrin in the heterodimeric entity was responsible for WNV entry into cells, antibody (against {alpha}v{beta}3 and {alpha}v{beta}5 integrin) blockage of WNV binding on virus overlay protein blot assay was performed. As shown in Fig. 2e, incubation of anti-{alpha}v{beta}3 integrin antibody (25 µg/ml) totally blocked the binding of WNV to the 105-kDa plasma membrane glycoprotein (lane 1) and to a much lesser extent, incubation of anti-{alpha}v{beta}5 integrin antibody (lane 3). Lane 2 represents WNV binding to the glycoprotein in the absence of antibody treatment. In addition, cells pre-incubated with anti-{alpha}v{beta}3 integrin antibody showed a drastic reduction (>70% at 25 µg/ml) in WNV entry into cells, whereas anti-{alpha}v{beta}5 integrin-pretreated cells showed partial inhibition (28% at 25 µg/ml) of WNV entry (Fig. 2f).

We have also repeated this set of experiments with other flaviviruses, such as JEV, under the same sero-complex as WNV) and DV (different sero-complex from WNV) (Fig. 2f). The inhibition pattern for JEV was similar to that of WNV. At the concentration of 25 µg/ml of anti-{alpha}v{beta}3 integrin antibody, entry of JEV into Vero cells was inhibited by 70% (Fig. 2f). Interestingly, the entry of DV into Vero cells was only partially blocked by anti-{alpha}v{beta}3 integrin antibody (Fig. 2f). Therefore, this suggests that {alpha}v{beta}3 integrin is specific in mediating the entry of WNV and JEV.

Soluble {alpha}v{beta}3 Integrin Blocks WNV Entry into Cells—To further confirm the specificity of {alpha}v{beta}3 integrin in mediating the entry of WNV into cells, we incubated soluble {alpha}v{beta}3 and {alpha}v{beta}5 integrin with WNV before overlaying this complex onto a Vero cell monolayer. Soluble {alpha}v{beta}3 integrin can effectively block WNV infection in a dose-dependent manner (at 10 µg/ml, there was >80% inhibition of virus infectivity), whereas soluble {alpha}v{beta}5 integrin showed minimal blockage of WNV infection (at 10 µg/ml, there was only <28% inhibition of virus infectivity), as shown in Fig. 3a. These results indicate the specific interaction of WNV with {alpha}v{beta}3 integrin, hence preventing the subsequent infection of Vero cells.



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  		FIG. 3.
Effect of soluble integrins and physiological ligands on WNV entry into host cells. a, dose-dependent inhibition of WNV infectivity by soluble {alpha}v{beta}3 integrin is clearly demonstrated (INTAVB3). Soluble {alpha}v{beta}5 integrin (INTAVB5) has minimal effect in blocking the infectivity of WNV. Results of three independent experiments (each data point represents the mean ± S.D. of results) are shown. b, entry of WNV is not significantly blocked by the binding of physiological ligands to integrins on the cell surface. Light shaded and dark shaded bars represent 50 µg/ml and 100 µg/ml ligands used, respectively. Results of three independent experiments are shown. FN, fibronectin; VN, vitronectin; LM, laminin; CS, chondroitin sulfate; HP, heparin; CL, collagen type 1; BSA, bovine serum albumin; RGD, Gly-Arg-Gly-Asp-Ser peptide; RGE, Gly-Arg-Gly-Glu-Ser. c, effects of EDTA (divalent cation chelator) on WNV and JEV binding to Vero cells. The removal of cations from the cell culture environment did not affect the binding of WNV and JEV to the cell surface of Vero cells.

 
Entry of WNV Is Not Highly Dependent on the RGD Motif—At the same time, we assayed for the effect of physiological integrin ligands (fibronectin, vitronectin, heparin, chondroitin sulfate, laminin, collagen type 1) and glycoprotein (BSA) on WNV entry into Vero cells. Results showed that the binding of fibronectin and vitronectin (both of which are known to interact with {alpha}v{beta}3 integrin by RGD-dependent motif) to the cell surface resulted in partial inhibition of WNV entry by 38 and 33% at 100 µg/ml, respectively (Fig. 3b). The rest of the ligands (heparin, chondroitin sulfate, and laminin) and glycoprotein (BSA), when incubated with Vero cells before virus inoculation, had minimal effect in preventing WNV entry into cells (Fig. 3b). Because the envelope protein of mosquito-borne flaviviruses (except DV) contain a RGD or RGE motif in the proposed receptor binding domain III (18), we further evaluated whether these motifs are responsible for binding to {alpha}v{beta}3 integrin. Vero cells were incubated with synthetic RGD peptides (Gly-Arg-Gly-Asp-Ser or Gly-Arg-Gly-Glu-Ser) before the inoculation of WNV. The RGD peptides exhibited partial blocking of WNV entry into Vero cells when high but non-cytotoxic concentrations were used (Fig. 3b).

In addition, the binding of physiological ligands to integrin often required the presence of divalent cations. The requirement of divalent cations for WNV binding to {alpha}v{beta}3 integrin was also investigated by pretreating cells with EDTA (divalent cation chelators) before virus infection. Vero cells treated with EDTA (at concentration -3 to 12 mM) did not block the binding and subsequent entry of WNV and JEV (Fig. 3c).

Gene Silencing of Human {beta}3 Integrin Inhibits WNV Entry into Cells—Next, we investigated the ability of cells with down-regulated cell surface expression of human {alpha}v{beta}3 integrin to mediate WNV infection. Gene knockout by means of siRNA technology was first carried out to down-regulate the expression of human {beta}3 integrin. siRNA against specific sequence encoding for human {beta}3 integrin was constitutively expressed from mammalian expression vector pSilencer 3.0-H1 (Ambion). Because the siRNA is designed to target only the sequence of human {beta}3 integrin, the recombinant plasmid were transfected into HeLa cells instead of Vero cells for this part of the study. Previous study has shown that the expression of {beta}3 integrin subunit is required for the proper targeting of {alpha}v subunit to the cell surface (20). Therefore, in the absence of {beta}3 subunit expression in the transfected cells, the heterodimeric complex of {alpha}v{beta}3 integrin will not be detected on the cell surface. Transfected cells were first screened for the down-regulation of cell surface and endogenous {alpha}v{beta}3 integrin expression. Fig. 4a shows the down-regulation of cell surface {alpha}v{beta}3 integrin when compared with mock-transfected cells (Fig. 4b) by immunofluorescence assay. HeLa cells with >80% of down-regulated expression of the {alpha}v{beta}3 integrin were selected by FACS for WNV entry assay (Fig. 4, c and d). The abolishment of {alpha}v{beta}3 integrin expression exhibited significant reduction of 60% of virus entry when compared with non-treated HeLa cells (Fig. 4e). Furthermore, there was no difference in the abolishment of virus entry for cells with down-regulated expression of glyceraldehyde-3-phosphate dehydrogenase (internal control) and pSilencer vector (Fig. 4e).



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  		FIG. 4.
Gene silencing of human {alpha}v{beta}3 integrin reduced WNV entry. a, down-regulated expression of human {alpha}v{beta}3 integrin in HeLa cells can also be observed by immunofluorescence assay as compared with non-transfected cells (without pSilencer-INTB3) (b). Similarly, HeLa cells were also incubated with anti-{alpha}v{beta}3 integrin antibody (transparent profiles) or mouse IgG2a control antibody (solid profiles) followed by staining with fluorescein isothiocyanate-labeled anti-mouse IgG. Down-regulated expression of {alpha}v{beta}3 integrin in HeLa cells transiently transfected with pSilencer-INTB3 was observed (c) when compared with the non-transfected cells (d) using flow cytometry analysis. e, drastic reduction of WNV entry into HeLa cells expressing siRNA against {alpha}v{beta}3 integrin can be observed. In contrast, entry of WNV into HeLa cells expressing siRNA against glyceraldehyde-3-phosphate dehydrogenase or transfected with siRNA expression vector (negative control) is not affected.

 
Expression of Recombinant {alpha}v{beta}3 Integrin Increased the Susceptibility of Cells to WNV Infection—A correlation analysis of the relative abundance of {alpha}v{beta}3 integrin expression on the surface of various target cells and their susceptibility to WNV infection were also assessed. The different cell lines were first incubated with antibodies against {alpha}v{beta}3 integrin followed by incubation with fluorescein isothiocyanate anti-mouse antibodies and examination by FACS. Different target cells (5 x 106 cells) were also infected with WNV at an m.o.i. of 10, and the productive virus yields were determined by plaque assays (expressed as plaque-forming units/ml). High level expression of {alpha}v{beta}3 integrin in Vero, baby hamster kidney, chick embryo fibroblast, HepG2, HeLa, and N2a cells were noted, and these cells were more susceptible to WNV infection (Table II) as compared with CS-1 (melanoma cells which did not synthesize the endogenous {beta}3 integrin subunit) and U937 (a monocytic cell line that did not express cell surface {alpha}v{beta}3 integrin) (Table II). Indeed, the expression of {alpha}v{beta}3 integrin in many different target cells supported WNV infection.


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  		TABLE II
Expression of {alpha}v{beta}3 integrin and permissivity to WNV infection

 
To affirm specifically the functional role of {alpha}v{beta}3 integrin in mediating WNV entry, we infected melanoma cells CS-1 (which did not synthesis endogenous {beta}3 integrin) and CS-1{beta}3 (which was capable of expressing recombinant {beta}3 integrin and forming functional {alpha}v{beta}3 integrin on the cell surface) (20) and quantitated the WNV entry. A minimal level of {alpha}v{beta}3 integrin cell surface expression was detected in CS-1 (Fig. 5a) as compared with CS-1{beta}3 cells (Fig. 5b). Virus entry assay was carried out to quantify the amount of WNV particles gaining entry into CS-1 or CS-1{beta}3 cells. Entry of WNV into CS-1 cells was ~5-fold lower than that of CS-1{beta}3 cells (Fig. 5c). Furthermore, a low virus titer of 5 x 102 plaque-forming units/ml was obtained at 4 days postinfection for CS-1-infected cells with minimal cytopathic effects (Fig. 5d). In contrast, total cytopathic effect was observed in infected CS-1{beta}3 cells within 22 h postinfection (Fig. 5d), and a productive virus yield of 8 x 107 plaque-forming units/ml of virus was obtained. Therefore, the expression of {alpha}v{beta}3 integrin had significantly promoted the susceptibility of CS-1 cells to WNV infection.



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  		FIG. 5.
Expression of {alpha}v{beta}3 integrin in CS-1 cells increased its permissiveness for WNV. Expression of {alpha}v{beta}3 integrin on the cell surface of CS-1 (a) and CS-1{beta}3 (b) cells was detected with anti-{alpha}v{beta}3 integrin antibody (transparent profile) or mouse IgG1 control antibody (solid profiles) followed by staining with fluorescein isothiocyanate-labeled anti-mouse IgG. The solid profile is overlapping exactly with the transparent profile. FACS was used to detect integrin expression. Cell surface expression of {alpha}v{beta}3 integrin is observed in CS-1{beta}3 cells but not CS-1 cells. c, entry of WNV is significantly enhanced in CS-1{beta}3 cells (compared with CS-1 cells) for the expression of {alpha}v{beta}3 integrin on the cell surface. d, obvious cytopathic effects are observed in WNV-infected CS-1{beta}3 cells within 22 h postinfection but not in virus-infected CS-1 cells. CPM, counts/min.

 
Activation of Integrin-associated Signaling Pathway by WNV—Because the engagement of physiological ligands to integrin is known to activate its associated outside-in signaling pathway, the ability of WNV infection to trigger integrin-associated outside-in signaling pathway was also determined. The complex interactions among ligands, integrins, numerous signaling molecules (including FAK, C-Src, p130cas) and cytoskeletal proteins (talin, paxillin, vinculin, and {alpha}-actinin) form aggregated patches (known as focal adhesion) on the plasma membrane (21).

Recent studies conducted by McLean et al. (22) and Akula et al. (23) have shown that the residue Tyr397 of FAK specifically undergoes autophosphorylation in response to the interaction of ligands/virus and integrin at the focal adhesion sites. The autophosphorylation of FAK is also responsible for the endocytosis signaling process. As such, the autophosphorylation of FAK at Tyr397 during WNV interaction with {alpha}v{beta}3 integrin in Vero cells was also investigated. Phosphorylation of FAK was detected with antibody specific for the phosphorylated form of FAK. Phosphorylated FAK was not detected in serum-starved cell lysate (Fig. 6a, lane 1). Induction of Vero cells with 200 ng/ml LPA (positive control) resulted in FAK phosphorylation within 5 min (Fig. 6a, lane 7) and was sustained over a period of 30 min (Fig. 6a, lane 8). Phosphorylation of FAK in WNV-infected cells was detected within 5 min after adding the virus to Vero cells (Fig. 6a, lane 2) and was sustained until 10 min after virus infection (Fig. 6a, lane 3). Dephosphorylation of FAK was observed as infection progressed from 20 to 45 min postinfection with WNV (Fig. 6a, lanes 4–6). To ensure that equal amounts of cellular proteins were loaded into each of the wells, the membrane was stripped and reprobed with antibody against actin. Equal quantities of actin were observed throughout the lanes (Fig. 6b).



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  		FIG. 6.
West Nile virus activates the integrin-dependent FAK. Activation of FAK autophosphorylation is via the engagement of WNV with {alpha}v{beta}3 integrin. a, West Nile virus induces phosphorylation of FAK. Lane 1, serum-starved Vero cells were not induced (negative control). Lane 2, Vero cells were infected with WNV for 5 min. Lane 3, Vero cells were infected with WNV for 10 min. Lane 4, Vero cells were infected with WNV for 20 min. Lane 5, Vero cells were infected with WNV for 30 min. Lane 6, Vero cells were infected with WNV for 45 min. Lane 7, Vero cells were induced with 200 ng/ml of LPA (positive control) for 5 min. Lane 8, Vero cells were induced with 200 ng/ml of LPA (positive control) for 30 min. Autophosphorylation of FAK is observed within 5 min of WNV infection and undergoes dephosphorylation after 30 min of WNV infection. b, the membrane is stripped and reprobed with antibody against actin to ensure equal amounts of cell lysate were loaded in each of the wells. Localization of FAK and vinculin in mock- and WNV-infected cells using immunofluorescence assay. c, FAK (green speckles) and vinculin (stained red) is not co-localized in serum-starved Vero cells. d, co-localization (yellow staining) of FAK and vinculin is observed in Vero cells infected with WNV for a period of 5 min. e, co-localization (yellow staining) of FAK and vinculin is also observed in Vero cells after treatment with 200 ng/ml of LPA (positive control).

 
During the process of outside-in signaling by integrins, localization of activated FAK (a non-receptor protein-tyrosine kinase) with vinculin at the focal adhesion was also observed (24). The distribution of vinculin and FAK in serum-starved (negative control) Vero cells, LPA activated Vero cells (positive control), and WNV-infected Vero cells were also analyzed by immunofluorescence assay. Both FAK and vinculin were observed to be more diffused and did not co-localize in serumstarved Vero cells (Fig. 6c). In contrast, a distinct patchy pattern of vinculin and FAK (co-localization of the two proteins) was observed in WNV-infected Vero cells (within 5 min of infection, Fig. 6d) and Vero cells treated with 30 ng/ml of LPA (Fig. 6e). These results demonstrated the activation of FAK, presumably upon the binding of WNV to {alpha}v{beta}3 integrin. Together, these results further suggested the specific interaction of WNV and {alpha}v{beta}3 integrin in mediating the activation of outside-in integrin-associated signaling pathway. Future work is required to further address the detailed process of the signaling pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
For enveloped viruses to gain entry into cells, the initial process involved complex interactions of viral attachment proteins with cellular receptor(s), co-receptor(s), or co-factor(s). These interactions are required to trigger downstream signals for the internalization of virus particles. For flaviviruses, the domain III of the envelope protein is proposed as the virus attachment protein. Monoclonal antibodies generated against domain III of the envelope protein are noted to be the strongest blockers of virus entry (9). Numerous neutralization epitopes for WNV have also been mapped within domain III of the envelope protein (25). Preliminary experiments performed to analyze the interaction of domain III of WNV envelope protein with {alpha}v{beta}3 integrin showed promising results of specific interaction between the cellular and virus structural proteins.2 However, we cannot exclude the possibility that other domains of the envelope protein may also participate in binding to {alpha}v{beta}3 integrin. In most mosquito-borne flaviviruses, domain III of the envelope protein contains an RGD motif or a very similar sequence (18), and these RGD motifs play essential roles in integrin-ligand interactions (26).

Several viruses, including foot-and-mouth disease virus (27), coxsackievirus (28), and adenovirus (29) were shown to bind to integrins in an RGD-dependent fashion. However, the binding of WNV to {alpha}v{beta}3 integrin does not specifically occur on the RGD binding site as illustrated with ligands of the integrin and RGD peptide blockage studies (Fig. 3b). The partial inhibition of WNV entry by fibronectin, vitronectin, and RGD peptides could probably be due to steric hindrance. The concentration of ligands and RGD peptides used in this study were also known to cause steric hindrance of other viruses binding to integrin (19). Furthermore, this result is also consistent with several reports that the RGD/RGE motif on the E protein of some flaviviruses is not essential in binding to the RGD motif binding site on integrins (18, 30). Mutagenesis of the RGD/RGE motif of yellow fever virus and Murray Valley encephalitis virus did not affect the absorption, penetration, and growth of the viruses (18, 30).

Because the entry of WNV and JEV can effectively be blocked by the antibody to the {alpha}v{beta}3 integrin (Fig. 2f), it is deduced that both WNV and JEV utilized {alpha}V{beta}3 integrin as a common receptor molecule in vertebrate cells. This is further supported by a recent study conducted by Volk et al. (31) showing the high structural similarity of the receptor binding region (domain III) on the envelope protein of both WNV and JEV. In contrast, the presence of structural differences in domain III of the DV envelope protein from that of WNV and JEV may explain the difference in receptor usage for entry. The cellular receptor(s) for virus entry could differ from one flavivirus to another. Dengue virus has been shown to bind to a number of cellular receptors, which include heparan sulfate (7), BiP (32), and dendritic cell-specific ICAM-3 grabbing non-integrin (15, 16). In contrast, dendritic cell-specific ICAM-3 grabbing non-integrin is not required for the entry of WNV and yellow fever virus into dendritic cells (15). However, it was also noted that the antibody against {alpha}v{beta}3 integrin did not completely block WNV entry into Vero cells, even in the presence of high concentrations of antibodies. It is plausible that additional co-factors may be involved in the entry process. Nevertheless, the binding of WNV to {alpha}v{beta}3 integrin is highly specific, and {alpha}v{beta}3 integrin is the main player in initiating virus entry.

The identification of {alpha}v{beta}3 integrin as the cellular receptor for WNV is of importance in understanding virus replication, pathogenesis, and tissue tropism in the host. The relatively high sequence conservation in the gene encoding for {alpha}v or {beta}3 integrin subunits may support the broad host range (invertebrate to vertebrate) of WNV infection (33). However, it is currently not known whether WNV also utilizes {alpha}v{beta}3 integrin or related molecules as the receptor for entry into mosquito cells. Work is currently being carried out to address this issue.

In a previous study by Chu and Ng (34), a high level of expression of the putative WNV receptor molecules at the apical surface of polarized Vero (C1008) cells was observed. These receptor molecules facilitate the preferential entry of WNV and kunjin virus (a subtype of WNV) through the apical surface. Similarly, apical localization of {alpha}v and {beta}3 integrin subunits expression was observed in polarized cells (35). This provided further evidence of the specificity of WNV for {alpha}v{beta}3 integrin. The detailed entry pathway of WNV into vertebrate cells was recently characterized in Ref. 36, in which the internalization of WNV occurred by clathrin-mediated endocytosis before translocating along the endolysosomal pathway for uncoating in a low pH environment. Joki-Korpela et al. (37) have also described the clathrin-mediated endocytosis of {alpha}v{beta}3 integrin as well as virus-{alpha}v{beta}3 integrin complexes. Furthermore, the endocytosis process of WNV is shown to have a heavy reliance on the actin filaments (36).

The entry pathway taken by WNV into Vero cells is consistent with the signaling pathway activated upon WNV binding to {alpha}v{beta}3 integrin in this study. The engagement of WNV with {alpha}v{beta}3 integrin triggered the activation of FAK (integrin-linked kinases), which is the central paradigm of outside-in signaling by integrin. The autophosphorylation of FAK in response to virus-integrin engagement leads to the formation of phosphotyrosine docking sites for several classes of signaling molecules. This is necessary for the recruitment and activation of the down-stream signaling molecules and signaling complexes that eventually lead to the triggering of actin assembly followed by the process of clathrin-mediated endocytosis (38, 39) of WNV particles.

To our knowledge, this is the first study that has identified {alpha}v{beta}3 integrin as the functional receptor for WNV and JEV in vertebrate cells. Future studies are required to ascertain the importance of the contribution of virus-receptor interaction in the pathogenesis of flavivirus infection in vivo. Nevertheless, the discovery of the functional receptor molecule has opened a new avenue of exploration into the use of prophylactic anti-virals against WNV and JEV infection. The interaction of virus-{alpha}v{beta}3 integrin can serve as a potential target for anti-viral strategies. The rational design of antagonists that block virus-{alpha}v{beta}3 integrin interaction may represent a novel concept to treat WNV and JEV infection.


   FOOTNOTES
 
* This work is supported by the Biomedical Research Council (Singapore), Project No. 01/1/21/18/003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 65-8743283; Fax: 65-7766872; E-mail: micngml{at}nus.edu.sg.

1 The abbreviations used are: WNV, West Nile virus; DV, dengue virus; BSA, bovine serum albumin; m.o.i., multiplicity of infection; FACS, fluorescence-activated cell sorter; FAK, focal adhesion kinase; LPA, lysophosphatidic acid; siRNA, small interfering RNA; JEV, Japanese encephalitis virus. Back

2 J. J.-H. Chu and M.-L. Ng, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Loy Boon Pheng for technical assistance and Dr. D. Cheresh (Scripps Research Institute) for providing the melanoma cell lines CS-1 and CS-1{beta}3.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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