Sialylated glycans as receptor and inhibitor of enterovirus 71 infection to DLD-1 intestinal cells - PubMed (original) (raw)

Sialylated glycans as receptor and inhibitor of enterovirus 71 infection to DLD-1 intestinal cells

Betsy Yang et al. Virol J. 2009.

Abstract

Background: Many viruses recognize specific sugar residues, particularly sulfated or sialylated glycans, as the infection receptors. A change of sialic acid (2-6)-linked galactose (SA-alpha2,6Gal) to SA-alpha2,3Gal determines the receptor for avian flu infection. The receptor for enterovirus 71 (EV71) infection that frequently causes fatal encephalitis in Asian children remains unclear. Currently, there is no effective vaccine or anti-virus agent for EV71 infection. Using DLD-1 intestinal cells, this study investigated whether SA-linked glycan on DLD-1 intestinal cells was a receptor for EV71, and whether natural SA-linked sugars from human milk could block EV71 infection.

Results: EV71 specifically infected DLD-1 intestinal cells but not K562 myeloid cells. Depletion of O-linked glycans or glycolipids, but not N-linked glycans, significantly decreased EV71 infection of DLD-1 cells. Pretreatment of DLD-1 cells with sialidase (10 mU, 2 hours) significantly reduced 20-fold EV71 replication (p < 0.01). Taken together, these results suggest that SA-linked O-glycans and glycolipids, but not N-glycans, on DLD-1 cells were responsible for EV71 infection. Purified SA-alpha2,3Gal and SA-alpha2,6Gal from human milk significantly inhibited EV71 infection of DLD-1 cells, indicating terminal SA-linked glycans could be receptors and inhibitors of EV71 infection.

Conclusion: This is the first in the literature to demonstrate that EV71 uses SA-linked glycans as receptors for infection, and natural SA-linked glycans from human milk can protect intestinal cells from EV71 infection. Further studies will test how a SA-containing glycan can prevent EV71 in the future.

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Figures

Figure 1

Figure 1

Infection of DLD-1 cells by EV71. EV71 infected and replicated in DLD-1 cells within 4 hours (4 H), and rapidly replicated in 24 hours (24 H) (A). The replication of EV71 in DLD-1 cells could be visible by specific antibody directed immunofluorescent staining (B). In contrast, EV71 did not infect K562 myeloid cells in 24 hours (C). Data presented are calculated from 4 experiments.

Figure 2

Figure 2

O-glycans and glycolipids but not N-glycans responsible for the EV71 infection. O-glycan synthesis inhibitor, benzyl N-acetyl-α-D-galactosaminide and glycolipid anchorage inhibitor, phosphotidylinositol-specific phospholipase, but not N-glycan synthesis inhibitor, tunicamycin, significantly inhibited EV71 infections. Data presented were calculated from three experiments.

Figure 3

Figure 3

Sialidase treatment of DLD-1 cells decreased EV71 infection. DLD-1 cells pre-treated with different doses of sialidase for 2 hours significantly reduced EV71 infection in 24 hours under immunofluorescent assay (upper panel). The treatment of sialidase at 10 mU/ml (P < 0.01) or at 50 mU/ml (P < 0.001) significantly inhibited EV71 infection of DLD-1 cells for 3 days as analyzed by qRT-PCR analysis of EV71 titers (lower panel). Data presented are derived from 4 experiments.

Figure 4

Figure 4

Inhibition of EV71 infection in DLD-1 cells by SA-α2,3Gal and SA-α2,6Gal. DLD-1 cells co-incubated with SA-linked galactose (SA-α2,3Gal or SA-α2,6Gal) significantly (P = 0.034) reduced the EV71 infection for 3 days as quantified by RT-PCR analysis. Data presented are calculated from 4 experiments.

Figure 5

Figure 5

Culture and quantification of EV71. EV71 were cultured in Vero cells at MOI = 2 until visible cytopathic effect (CPE) (A), and subjected to qRT-PCR analysis of virus titer (B). Based on qRT-PCR detection of a series of well-known EV71 titers (C), the virus titers were determined by an interpolation on the standard curve (D).

Figure 6

Figure 6

A proposed SA-based "double-edge sword" on blocking and destructioing of EV71 infections. SA (α2,6)-linked galactose can block EV71 infection by competition of sugar receptor, and the galactose can be linked with cationic compounds such as lactoferrin or chitosan for destruction of EV71.

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