Abstract

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD), a common febrile disease occurring mainly in young children. Although clinical manifestations of HFMD are usually mild and self limiting, a severe EV71 outbreak can lead to a diverse array of neurological diseases. Identification of the specific cellular receptors is crucial for elucidating the mechanism of early virus-host interactions and the pathogenesis of enteroviruses¹. Here we identify human P-selectin glycoprotein ligand-1 (PSGL-1; CD162), a sialomucin membrane protein expressed on leukocytes that has a major role in early stages of inflammation^{2, 3, 4}, as a functional receptor for EV71 using an expression cloning method by panning⁵. The N-terminal region of PSGL-1 binds specifically to EV71. Stable PSGL-1 expression allowed EV71 entry and replication, and development of cytopathic effects in nonsusceptible mouse L929 cells. Five out of eight EV71 strains bound soluble PSGL-1 and used intact PSGL-1 as the primary receptor for infection of Jurkat T cells. Three other EV71 strains did not use PSGL-1, suggesting the presence of strain-specific replication of EV71 in leukocytes. EV71 replicated in nonleukocyte cell lines in a PSGL-1–independent manner, indicating the presence of alternative receptor(s) for EV71. The identification of PSGL-1 as a receptor for EV71 sheds new light on a role for PSGL-1–positive leukocytes in cell tropism and pathogenesis during the course of HFMD and other EV71-mediated diseases.

Introduction

EV71 and coxsackievirus A16 (CVA16) belong to the human enterovirus species A of the genus Enterovirus⁶ and are the major causative agents of HFMD. EV71 may cause various neurological diseases, such as aseptic meningitis, acute flaccid paralysis and fatal neurogenic pulmonary edema. Severe EV71 outbreaks have been reported periodically throughout the world, representing a major public health concern, particularly in the Asia-Pacific region^{7, 8, 9}. During large outbreaks of EV71, individuals with severe EV71-associated encephalitis and neurogenic pulmonary edema showed a marked depletion of T cells and high levels of proinflammatory cytokines^{10, 11}. Because of this T cell involvement, we generated a retroviral complementary DNA (cDNA) library from Jurkat T cells that are susceptible to EV71 infection¹² and used it for expression cloning⁵ to identify a receptor that specifically binds EV71 virions (EV71-1095 strain, Supplementary Table 1). Transduction of mouse myeloma P3X63Ag8U.1 (P3U1) cells with the Jurkat cDNA library resulted in the formation of four colonies that bound EV71-1095–coated dishes, all of which encoded human PSGL-1 (SELPLG) (Fig. 1a and Supplementary Fig. 1).

Figure 1: EV71-1095 binds human PSGL-1 expressed on 293T cells.

(a) Schematic structure of human PSGL-1. The binding sites of PSGL-1–specific mAbs (KPL1 (ref. 14) and PL2 (ref. 15)) are indicated. aa, amino acids. (b) Flow cytometric analysis of 293T cells transfected with pEF-PSGL-1 or pEF (a control plasmid). Left, shaded and open areas represent staining with KPL1 mAb and isotype control, respectively. Right, shaded and open areas represent EV71-1095 binding assay and binding control with the mock-infected culture supernatant, respectively. The percentage of PSGL-1– or EV71-positive cells is indicated (mean s.d., n = 3). (c) EV71-1095 binding inhibition assay by flow cytometry. Dose-dependent inhibition of EV71-1095 binding to 293T cells transiently expressing PSGL-1 by mAb.

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To confirm the specific binding of PSGL-1 to EV71-1095, we used 293T cells, which express undetectable amounts of endogenous PSGL-1. Transient expression of human PSGL-1 on 293T cells markedly increased the binding of EV71-1095 to 293T cells (Fig. 1b); however, expression of control ligands (sialomucin proteins CD34 and CD43) and mouse Psgl-1 did not (Supplementary Fig. 2). To identify the region of human PSGL-1 that is important for EV71 binding, we first constructed a chimeric PSGL-1 (hmPSGL-1) containing amino acids 1–61 of the human PSGL-1 followed by amino acids 63–397 of mouse Psgl-1 (Supplementary Fig. 3a,b). EV71-1095 bound hmPSGL-1 expressed on 293T cells (Supplementary Fig. 3c). To confirm this finding, we examined whether monoclonal antibodies (mAbs) recognizing PSGL-1 (KPL1 (refs. 13,14) and PL2 (ref. 15); Fig. 1a) block the PSGL-1–EV71 interaction. KPL1, which blocks P-selectin binding to PSGL-1 (ref. 13), inhibited EV71-1095 binding to 293T cells transiently expressing PSGL-1 in a dose-dependent manner; however, PL2, which does not block binding to P-selectin, did not (Fig. 1c). These findings suggest that the N-terminal region (amino acids 42–61) of human PSGL-1 is crucial for interactions with EV71-1095.

To test whether PSGL-1 is involved in the later steps of viral entry after binding to cells, we used mouse cells that do not support EV71 infection. Mouse myeloma P3U1 cells expressing human PSGL-1 did not support efficient EV71 replication (data not shown). Therefore, we obtained an L929 cell clone (L-PSGL-1.1) that stably expresses high amounts of human PSGL-1 (Fig. 2a). L-PSGL-1.1 cells showed a cytopathic effect 4 d after infection, and we detected EV71 antigen by immunofluorescence in the infected L-PSGL-1.1 cells (Fig. 2b). The development of cytopathic effect and detection of viral antigens induced by EV71-1095 infection was markedly inhibited by KPL1 (Fig. 2b), but not by PL2 (data not shown), as were the replication kinetics of EV71-1095 in infected L-PSGL-1.1 cells (Fig. 2c,d), indicating PSGL-1–dependent EV71 replication in these cells. Furthermore, pretreatment of EV71-1095 with a soluble form of recombinant PSGL-1 (PSGL-1–Fc) impaired viral replication 2–4 d after viral inoculation of L-PSGL-1.1 cells (Fig. 2e) and RD cells (Supplementary Table 2) expressing undetectable PSGL-1 (data not shown), suggesting that the inhibitory effect of PSGL-1–Fc is due to either direct binding to EV71 virions or virion uncoating induced by the PSGL-1–Fc–EV71 interaction.

Figure 2: Stable expression of human PSGL-1 on mouse L929 cells permits infection by EV71-1095.

(a) PSGL-1 expression on L-PSGL-1.1 and L-bsd (PSGL-1 negative control) cells, as measured by flow cytometry. (b) Development of cytopathic effect (Phase) and EV71 antigens by immunofluorescence (VP1). Scale bars, 100 m. (c) Replication kinetics of EV71-1095 in L-PSGL-1.1 and PSGL-1–negative cells. (d) Replication kinetics of EV71-1095 in L-PSGL-1.1 cells in the presence of PSGL-1–specific (KPL1 and PL2) and control mAbs. (e) Replication kinetics of EV71-1095 in L-PSGL-1.1 cells after treatment with soluble PSGL-1. Viral titers and error bars are indicated as the means s.d. of triplicate analyses. Asterisks indicate P < 0.01 compared to the two controls.

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To elucidate the biological role of PSGL-1–dependent cell tropism of EV71-1095, we investigated the relationship between PSGL-1 expression on the cell surface (Fig. 3a) and replication of EV71 (Fig. 3b) in various cell lines (Supplementary Table 2). Among the four leukocyte cell lines examined, we found PSGL-1–dependent viral replication, as indicated by the reduction of viral titers in the presence of KPL1 in Jurkat T cells and U937 monocytic cells, which express large amounts of PSGL-1 on the cell surface. EV71-1095 induced a faint cytopathic effect in Jurkat T cells but no apparent cytopathic effect in the other leukocyte cell lines (data not shown). EV71-1095 replication was not affected by KPL1 in MT-2 cells and four nonleukocyte cell lines (RD, HEp-2c, SK-N-MC and Vero) that have little or no PSGL-1 expression, suggesting PSGL-1–independent replication through unidentified receptors in MT-2 and nonleukocyte cells. Taken together, these results suggest that EV71-1095 can use PSGL-1 as a functional cellular receptor in PSGL-1–positive leukocytes (Supplementary Fig. 1a), but it may use alternative mechanism(s) for replication in cells that have little or no PSGL-1 expression (Supplementary Fig. 1b).

Figure 3: PSGL-1 expression and EV71-1095 replication kinetics in leukocyte and nonleukocyte cell lines.

(a) PSGL-1 expression on the cell surface, as measured by flow cytometry. The shaded and open areas represent staining with PSGL-1–specific mAb (KPL1) and isotype control, respectively. (b) EV71-1095 replication kinetics in the presence of KPL1 and control mAbs. The titers and error bars are the means s.d. of triplicate analyses. Asterisks indicate P < 0.01 compared to the two controls.

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Although the association of a specific genogroup with severe neurological diseases has not been identified through molecular epidemiological analyses of previous and recent EV71 isolates (genogroups A, B and C)^{9, 16}, in vitro and in vivo phenotypes may be associated with certain amino acid determinants of EV71. Using eight representative EV71 strains (Supplementary Table 1), we investigated the strain specificity of EV71 and PSGL-1 use. We first examined the direct biochemical interaction between EV71 strains and PSGL-1–Fc by co-immunoprecipitation. The VP1 proteins of the SK-EV006, C7/Osaka, KED005, 1095 and 75-Yamagata strains of EV71 immunoprecipitated with PSGL-1–Fc (Fig. 4a). In contrast, the VP1 proteins of the BrCr, Nagoya and 02363 strains of EV71 did not immunoprecipitate with PSGL-1–Fc (Fig. 4a). Thus, these EV71 strains can be classified into two distinct phenotypes according to their PSGL-1–binding capability, regardless of genogroup: five PSGL-1–binding strains (EV71-PB; SK-EV006 (genogroup B3), C7/Osaka (B4), KED005 (C1), 1095 (C2) and 75-Yamagata (C4)) and three PSGL-1–nonbinding strains (EV71–non-PB; BrCr (A), Nagoya (B1) and 02363 (C1)). We then tested the PSGL-1–dependent replication competence of EV71-PB and EV71–non-PB strains in Jurkat T cells (Fig. 4b). All five EV71-PB strains replicated in Jurkat T cells in a PSGL-1–dependent manner, as indicated by the reduction of viral titers in the presence of KPL1 at 4 d after inoculation (Fig. 4b). Among the three EV71–non-PB strains, two (Nagoya and 02363) replicated in Jurkat T cells in a PSGL-1–independent manner, and the BrCr strain replicated poorly (Fig. 4b). These data indicate that EV71-PB strains use PSGL-1 as the primary and functional receptor for infection of Jurkat T cells (Supplementary Fig. 1a), whereas some of the EV71–non-PB strains may use unidentified receptors for viral replication even in PSGL-1–positive T lymphocytes (Supplementary Fig. 1c). Although the KED005 and 02363 strains (genogroup C1) show distinct differences in their PSGL-1–binding phenotypes, only four amino acids (Ile55 of VP3 and Lys98, Glu145 and Ile262 of VP1 for the EV71-02363 strain) are different in the proteins comprising the entire capsid region of these two strains, suggesting that a few amino acid determinants in the capsid proteins are fully responsible for the phenotype (Fig. 4). Among the four amino acid differences, Glu145 of VP1 may be exposed on a surface-exposed loop in the capsid VP1 protein, and this has been identified as a major virulence determinant in mouse models^{17, 18}.

Figure 4: EV71 strain–specific binding to PSGL-1 and replication in Jurkat T cells.

The strains that are EV71-PB and EV71–non-PB are indicated in red and blue, respectively. (a) Western blot showing the EV71 VP1 protein immunoprecipitated with PSGL-1–Fc. Concentrated viruses (lane 1) were incubated with isotype control mAb (lane 2), EV71-VP1–specific mAb (MA105, lane 3), negative control recombinant protein (CTLA4-Fc, lane 4) or PSGL-1–Fc (lane 5) and immunoprecipitated. (b) Viral replication in Jurkat T cells incubated with PSGL-1–specific (KPL1 and PL2) and control mAbs. Titers are expressed as the mean, and error bars indicate s.d. of triplicate analyses.

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We next investigated PSGL-1–dependent replication of the prototype CVA16-G10 strain. This strain replicated in L-PSGL-1.1 cells but not in L-bsd cells (a PSGL-1–negative control), and virus replication in L-PSGL-1.1 cells was inhibited by KPL1 (Supplementary Fig. 4a,b). The CVA16-G10 strain used PSGL-1 to infect L-PSGL-1.1 cells but not Jurkat T cells (Supplementary Fig. 4c), suggesting that CVA16-G10 represents a strain that uses an alternative entry mechanism via unidentified functional receptors for infection of Jurkat T cells.

PSGL-1 expressed on leukocytes has a crucial role in the tethering and rolling of leukocytes during recruitment of cells from blood vessels to the sites of acute inflammation upon stimulation by infection^{2, 3, 4}. The tissue expression of PSGL-1 is primarily restricted to hematopoietic myeloid, lymphoid and dendritic lineages and to platelets. However, PSGL-1 is also expressed on the dendritic cells of lymph nodes and on macrophages in the intestinal mucosa², which could be the primary site of EV71 replication after viral ingestion. During the viremic phase of infection, a variety of circulating leukocytes expressing PSGL-1 may be responsible for the in vivo replication of EV71 (ref. 19) and subsequent EV71-induced apoptosis in the infected cells^{12, 20}, possibly resulting in the T cell depletion and changes in cytokine levels observed in severe encephalitis cases with pulmonary edema^{10, 11}. In addition, the distribution and recruitment of PSGL-1–positive Langerhans cells and lymphocytes in inflamed skin² are consistent with the apparent HFMD pathogenesis associated with EV71 and CVA16 infection, which is characterized by acute skin inflammation. In this regard, our findings suggest the involvement of PSGL-1–positive inflammatory cells during the course of EV71 and CVA16 infection.

Although EV71 infects neurons and causes acute brainstem encephalitis, paralysis or both in humans^{21, 22} and experimental animals^{17, 23, 24, 25}, PSGL-1 is apparently not expressed in the adult human brain². Consistent with this observation, we have shown that EV71-1095 may also use PSGL-1–independent mechanism(s) for replication in nonleukocyte cells, including SK-N-MC neuroblastoma cells. Furthermore, CVA16, another causative agent of HFMD, can also use PSGL-1 as a functional receptor in L-PSGL-1.1 cells. Thus, PSGL-1–dependent viral replication is unlikely to be directly responsible for the neuronal cell apoptosis induced by EV71 (refs. 26,27). However, we cannot exclude the possible involvement of PSGL-1–positive inflammatory cells in the pathogenesis of HFMD and a variety of EV71 diseases with or without neurological manifestations.

We have shown that human PSGL-1 is a functional cellular receptor for EV71 infection. The occurrence of severe EV71 infection with a number of fatal encephalitis cases continues to be a major public health threat even now²⁸, but, currently, no vaccines or specific antiviral agents are available for EV71. Because soluble PSGL-1 inhibits EV71 replication, it may be a potential inhibitor of EV71-PB infection. The identification of PSGL-1 as a functional EV71 receptor on leukocytes is the first major step in elucidating EV71 pathogenesis at the molecular level. However, other EV71 and human enterovirus species A receptors on leukocyte and nonleukocyte cells may also have key implications in EV71 tropism and pathogenesis, particularly for severe central nervous system diseases. Further structural and functional analyses of interactions of EV71 with PSGL-1 and other unidentified receptors may provide new therapeutic approaches for the treatment of severe EV71 diseases.

Note: Supplementary information is available on the Nature Medicine website.

Acknowledgments

We thank N. Takeda, S. Morikawa, Y. Matsuura, K. Moriishi, S. Koike, S. Yamayoshi and Y. Izumiya for helpful discussions; Y. Ami for technical advice regarding FACS; and N. Nishimura for preparing figures. We also thank M. Sinniah, M. Yusof (Institute for Medical Research, Malaysia) for providing EV71-SK-EV006 and KED005, N. Onnimala, Y. Pongsuwanna (National Institute of Health, Thailand) for providing EV71-02363, Y. Okuno (Osaka Prefectural Institute of Public Health) for providing EV71-C7/Osaka, K. Mizuta (Yamagata Prefectural Institute of Public Health) for providing EV71-75-Yamagata, A. Makino, Y. Tohya, H. Akashi (University of Tokyo) for providing P3U1 cells, H. Sakata (National Institute of Infectious Diseases, Japan) for providing L929 cells, and H. Shirato (National Institute of Infectious Diseases, Japan) for providing MOLT-4 and MT-2 cells. We are grateful to J. Wada for technical assistance. This work was supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Y.N.). Y.N. and H.S. were supported in part by a Grant-in-Aid for Research on Emerging and Re-emerging Infectious Diseases and a Grant-in-Aid for the Promotion of Polio Eradication, from the Ministry of Health, Labour and Welfare, Japan.

Author Contributions

Y.N. designed and performed experiments, analyzed data and wrote the paper; M.S. improved the expression cloning method; Y.T. prepared and characterized EV71-specific mAbs; and T.M. and T.W. analyzed data and wrote the paper. H.S. planned the project, designed experiments, analyzed data and wrote the paper. All authors discussed the results and commented on the manuscript.

Received 2 December 2008; Accepted 6 April 2009; Published online 21 June 2009.

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Online methods

Cells. L929 cells were generously provided by H. Sakata P3U1 cells were generously provided by H. Akashi. 293T cells were generously provided by Y. Matsuura. GP2-293 cells were obtained from Clontech. Jurkat cells were obtained from Riken Cell Bank. U937 cells were obtained from the Japanese Collection of Research Biosources. MOLT-4 and MT-2 cells were generously provided by H. Shirato. RD cells were obtained from the US Centers for Disease Control. HEp-2 cells were obtained from the Victorian Infectious Diseases Reference Laboratory. SK-N-MC cells were obtained from DS Pharma Biomedical. Vero cells were obtained from Japan Poliomyelitis Research Institute. We cultured L929, 293T and GP2-293 cells in DMEM (Sigma) supplemented with 10% FCS. We maintained P3U1, Jurkat, U937, MOLT-4 and MT-2 cells in RPMI-1640 medium (Sigma) with 10% FCS. We maintained RD, HEp-2c, SK-N-MC and Vero cells in Eagle's minimal essential medium (Nissui) supplemented with 10% FCS.

Viruses. We propagated all EV71 strains (Supplementary Table 1) in RD or Vero cells. Because some of the strains produced diffuse plaques on RD cells, we determined the viral titers by a microtitration assay using 96-well plates and RD cells as previously described²³. Briefly, we used ten wells for each viral dilution and expressed the viral titers as 50% cell culture infectious dose (CCID₅₀).

Monoclonal antibodies and recombinant proteins. We generated the EV71-specific mAbs MA105 (mouse IgG2b) and MA35 (mouse IgG2a) from mice immunized with EV71-1095 (Y. Tano et al., unpublished data). We purchased the EV71-specific mAb 10F0 from Biogenesis. We purchased the mAbs to human CD34 (clone 563), human CD43 (L60), human PSGL-1 (KPL1), and mouse Psgl-1 (4RA10) from BD Biosciences, and we purchased human PSGL-1–specific mAb PL2 from Beckman-Coulter. For negative controls, we purchased human (MOPC-21) and rat IgG1 (R3-34) and human IgG2b (27-35) from BD Biosciences. We used NaN₃-free mAbs, MOPC-21 (BioLegend) and NCG01 (Lab Vision) in cell culture as negative controls. We purchased soluble recombinant forms of human proteins fused to the Fc region of human IgG1 (PSGL-1–Fc and cytotoxic T lymphocyte antigen-4–Fc) from R&D Systems.

Preparation of EV71-1095–coated dishes and selection of Jurkat T cell clones. We incubated a polystyrene Petri dish (Ina-optika) with 10 g ml^-1 MA35 in 10 ml of 50 mM Tris-HCl (pH 9.4) at 4 °C overnight. We rinsed the dishes twice with PBS containing 2% FCS (PBS-2FCS) and blocked them with PBS-2FCS at 25 °C for 30 min. We then applied EV71-1095 (10 ml, 1 10^8.6 CCID₅₀ ml^-1) to the MA35-coated dishes and incubated at 25 °C for 30 min. We replaced the supernatant with a fresh viral preparation, and we repeated replacing the supernatant three times. We fixed the dishes with a viral preparation containing 1% paraformaldehyde for 30 min at 25 °C. Finally, we washed the dishes with PBS-2FCS five times and used them for selection and expression cloning.

We selected Jurkat T cell clones that expressed high levels of EV71-binding molecules on the cell surface with the panning method with EV71-1095–coated dishes. We added Jurkat T cells to the EV71-coated dishes in RPMI-1640 medium and incubated them at 4 °C for 90 min. We removed nonadherent cells by washing with RPMI-1640 medium, and we cultured the adherent cells for a week. We used the Jurkat T cell colonies on the EV71-coated dishes for preparation of the cDNA library.

Retroviral cDNA library. Using the Jurkat T cell colonies on the EV71-coated dishes, we prepared a retroviral cDNA library as previously described²⁹ with minor modifications. We used the Pantropic Retroviral Expression System (Clontech) to prepare vesicular stomatitis virus G protein–pseudotyped retroviruses. We ligated the Jurkat cDNA with the EcoRI adaptor and cloned it into the EcoRI site of the pLIB retroviral vector (Clontech). We transfected the pLIB plasmid along with a vesicular stomatitis virus G protein expression plasmid into GP2-293 cells with Lipofectamine 2000 (Invitrogen). We harvested the culture supernatant 2 d after transfection and used it to infect P3U1 cells.

Expression cloning of the EV71-binding receptor. We preformed retrovirus-mediated expression cloning using EV71-coated dishes and P3U1 cells as described previously⁵. We isolated genomic DNA from each P3U1 cell colony bound to the EV71-coated dishes, subjected it to PCR amplification with pLIB-specific primers and sequenced the amplification product.

Expression plasmids. We cloned the cDNAs encoding human SELPLG, CD34, SPN and mouse Selplg into pEF6-Flag-3S to produce pEF-PSGL-1, pEF-CD34, pEF-CD43 and pEF-mPsgl-1, respectively. For hmPSGL-1 expression, we constructed chimeric SELPLG cDNA by PCR and cloned it into pEF6-Flag-3S. The primers used for cDNA cloning and chimeric protein construction and mutation are shown in Supplementary Tables 3 and 4. More details are provided in the Supplementary Methods.

Detection of cell surface molecules by flow cytometry. We washed cells once with flow cytometry buffer (PBS supplemented with 2 mM EDTA, 2% FCS and 0.1% NaN₃) and incubated them with mAb on ice for 30 min. After washing with flow cytometry buffer, we incubated the cells with secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen). We washed and analyzed the cells by FACSCalibur (BD Biosciences).

EV71-binding assay by flow cytometry. We collected 293T cells 24 h after transfection with expression plasmids, washed them once with flow cytometry buffer and incubated them with the EV71-1095 preparation (1 10⁷ CCID₅₀) supplemented with 0.1% NaN₃ or concentrated viruses (containing 0.5 g of VP1 protein) per 50 l flow cytometry buffer. In the binding inhibition assay with mAbs, we treated cells with mAb for 30 min on ice before exposing them to the virus. We washed the cells and stained them with Alexa Fluor 488–conjugated MA105, washed them and then analyzed them by FACSCalibur.

Establishment of the L-PSGL-1.1 cell line. To establish a mouse L929 cell line expressing human PSGL-1, we transfected L929 cells with pEF-PSGL-1 and selected stable transfectants with 5 g ml^-1 blasticidin S HCl (Invitrogen). Through a limiting dilution of the various cell colonies, we selected three of the 16 cell lines on the basis of high PSGL-1 expression. Finally, we established L-PSGL-1.1 as the single cell clone that supported the most efficient EV71-1095 replication. As a PSGL-1–negative control, we also established an L-bsd cell line by transfecting L929 cells with pEF6-Flag-3S followed by selection with 5 g ml^-1 blasticidin.

Immunofluorescence microscopy. We fixed cells with 4% paraformaldehyde in PBS for 30 min at 25 °C, washed them, permeabilized them with PBS containing 1% Triton X-100 for 10 min at 25 °C, blocked them with PBS containing 1% BSA for 10 min at 25 °C and incubated them with VP1-specific mAb 10F0 labeled with Alexa Fluor 488 for 30 min at 37 °C. After a final wash, we analyzed the cells with a fluorescence microscope (Keyence).

Immunoprecipitation and western blotting. We diluted concentrated viruses (0.5 g VP1 protein) in 300 l of immunoprecipitation buffer (20 mM Tris-Cl, 135 mM NaCl, 1% Triton X-100 and 10% glycerol; pH 7.4) and incubated them with 1 g of mAb or chimeric proteins for 2 h at 4 °C. We added Dynabeads Protein G (Invitrogen) and incubated the mixture for an additional 2 h. We washed the beads and subjected the immunoprecipitates to 12.5% SDS-PAGE. For western blotting, we transferred the proteins onto nitrocellulose membranes and blotted with MA105.

Viral infection assays. We inoculated cells (4 10⁴ cells per 200 l in a 48-well plate) with viruses at 10 CCID₅₀ per cell for 1 h, washed them and incubated them in medium at 34 °C (for L-PSGL-1.1, Jurkat, U937, MOLT-4 or MT-2 cells) or 37 °C (for RD, HEp-2c, SK-N-MC or Vero cells). For mAb inhibition, we pretreated the cells with 10 g ml^-1 mAb for 1 h, washed them and maintained them in the medium with 10 g ml^-1 mAb. For inhibition with PSGL-1–Fc, we pretreated EV71-1095 (1 10⁵ CCID₅₀) with 1 g PSGL-1–Fc per 100 l for 1 h and then inoculated them into L-PSGL-1.1 cells. We incubated the cells for 1 h in the presence of PSGL-1–Fc, washed them and maintained them in the absence of PSGL-1–Fc. We subjected the culture supernatants and infected cells to three cycles of freeze-thawing before titration.

Statistical analyses. We carried out all infection assays in triplicate and compared the mean viral titers with Student's t test (two-tailed). We considered P values of <0.01 statistically significant.

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