Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor - PubMed (original) (raw)

Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor

Chee Wah Tan et al. J Virol. 2013 Jan.

Abstract

Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor.

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Figures

Fig 1

Inhibitory effects of GAGs and inhibitors. (A) For the viral inactivation assay, various concentrations of GAGs, polyanionic dextran sulfate, and suramin were preincubated with EV-71 particles for 1 h at 37°C before infection of RD cells at room temperature. (B and C) For the cell protection assay, various concentrations of GAGs (B) or of poly-

-lysine or anti-heparan sulfate peptides, designated peptides G1 and G2 (C), were preincubated with RD cells for 1 h at 37°C before EV-71 infection. The viral RNA was extracted and quantified by quantitative real-time PCR 24 h postinfection.

Fig 2

Inhibitory effects of heparin on EV-71 isolates and the PV vaccine strain. EV-71 and PV particles were pretreated with heparin at a final concentration of 2,500 μg/ml for 1 h at 37°C before infection of RD cells. The low-passage-number EV-71 isolates (14716, 35017, 1657640, and 1687413) and the PV vaccine strain were obtained from the Diagnostic Virology Laboratory, University Malaya Medical Center. The titers of EV-71 and the PV vaccine strain were quantified 24 h postinfection by TaqMan quantitative real-time PCR and plaque assays, respectively.

Fig 3

(A) EV-71 was preincubated with various concentrations of heparin and desulfated heparin variants for 1 h at 37°C before infection of RD cells. (B) Inhibitory effect of sodium chlorate on different EV-71 strains in RD cells. RD cells were pretreated with increasing concentrations (0 mM, 10 mM, 30 mM, and 50 mM) of sodium chlorate for 24 h before EV-71 infection at an MOI of 0.1. (C) Transient siRNA knockdown of NDST-1 and EXT-1 expression. NDST-1, EXT-1, and negative-control siRNAs in the Lipofectamine 2000 reagent were transfected into RD cells for 24 h before EV-71 infection. The viral load was determined 24 h postinfection by TaqMan quantitative real-time PCR.

Fig 4

Treatment of RD cells with heparinase or chondroitinase ABC. (A) Inhibitory effects of heparinase I/II/III and chondroitinase ABC on EV-71 infection. RD cells were pretreated with heparinase or chondroitinase ABC for 1 h at 37°C before EV-71 infection at an MOI of 0.1. The viral RNA was extracted and evaluated by TaqMan quantitative real-time PCR. (B) Inhibitory effect of heparinase I on different EV-71 strains and the PV vaccine strain. (C) Confocal microscopy analysis (with a 40× objective) of EV-71 binding assays after heparinase I or chondroitinase ABC treatment. EV-71 particles were stained with a monoclonal anti-EV-71 antibody and were subsequently stained with Alexa Fluor 488-labeled anti-mouse IgG. Nuclei were stained with DAPI. EV-71 particles and nuclei are shown in green and blue, respectively.

Fig 5

Binding of EV-71 to CHO-K1 and CHO mutant cells. CHO-K1 cells and cells of CHO mutants defective in proteoglycan synthesis were assessed for their abilities to bind to EV-71. The CHO-pgsA745 cell line lacks heparan sulfate and chondroitin sulfate proteoglycans, while the CHO-pgsD677 cell line lacks heparan sulfate proteoglycan but produces 15% of normal proteoglycans. The binding of EV-71 to parental and mutant CHO cells was assayed by using confocal microscopic analysis (A) and a Cellomics ArrayScan VTI HCS reader with Spot Detector BioApplication software (B) and was verified by TaqMan quantitative real-time PCR.

Fig 6

Binding of EV-71 and PV to an immobilized heparin-Sepharose column. EV-71 and PV supernatants were passed through a column of immobilized heparin-Sepharose and were eluted with 2 M NaCl. The titers of EV-71 and the PV vaccine strain from each fraction were quantified by TaqMan quantitative real-time PCR and plaque assays, respectively. EV-71 levels are presented as viral RNA copies per milliliter, and levels of the PV vaccine strain are presented as PFU per milliliter. Error bars represent means ± SD for each fraction.

Fig 7

Three-dimensional pentameric structure and sequence alignment of EV-71. The structure of the EV-71 pentamer was generated using the DeepView-Swiss PDB viewer. The molecular structures of EV-71 VP1, VP2, VP3, and VP4 are presented in blue, green, red, and purple, respectively. The amino acids Arg166, Lys242, and Lys244 are presented in yellow, white, and light blue, respectively. (A) Top view of the EV-71 pentamer. (B) Side view of the EV-71 pentamer. (C) Histogram showing sequence consensus in the VP1 region of EV-71. A total of 174 sequences were aligned and analyzed using ClustalW2. The arbitrary scale is shown at the left of the histogram; 1.0 denotes perfect consensus at a given amino acid site across all entries. The alignments of Arg166, Lys242, and Lys244 in representative EV-71 strains from genotypes A, B, and C are shown above the histogram.

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Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor - PubMed (original) (raw)